EP2820424B1 - Soluble manf in pancreatic beta-cell disorders - Google Patents

Soluble manf in pancreatic beta-cell disorders Download PDF

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EP2820424B1
EP2820424B1 EP13741203.7A EP13741203A EP2820424B1 EP 2820424 B1 EP2820424 B1 EP 2820424B1 EP 13741203 A EP13741203 A EP 13741203A EP 2820424 B1 EP2820424 B1 EP 2820424B1
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pancreatic
cell
subject
soluble manf
cells
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EP2820424A4 (en
EP2820424A1 (en
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Fumihiko Urano
Kohsuke KANEKURA
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University of Massachusetts UMass
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • A61K31/47—Quinolines; Isoquinolines
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    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/507—Pancreatic cells
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    • C12N2310/00—Structure or type of the nucleic acid
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    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • pancreatic ⁇ -cells Loss of the function or number of pancreatic ⁇ -cells in a subject contributes to the pathogenesis of several diseases, including type 1 diabetes (diabetes mellitus), type 2 diabetes, and Wolfram syndrome.
  • type 1 diabetes the patient has high blood glucose levels because of insulin deficiency.
  • absolute deficiency of insulin occurs in patients with type 1 diabetes
  • relative deficiency of insulin occurs in patients with type 2 diabetes.
  • Increasing evidence indicates that reduced functional pancreatic ⁇ -cell mass is a common feature of both type 1 and type 2 diabetes, as well as genetic forms of diabetes such as Wolfram syndrome ( Pipeleers et al., Novartis Found Symp. 292:19-24, 2008 ).
  • pancreatic ⁇ -cell function and mass gradually decline, eventually leading to insulin deficiency and hyperglycemia.
  • pancreatic ⁇ -cells are susceptible to dysfunction and death ( Oslowski et al., Curr. Opin. Endocrinol. Diabetes Obes. 17:107-112, 2010 ; Oslowski et al., Curr. Opin. Cell Biol. 23:207-215, 2011 ; Fonseca et al., Trends Endocrinol. Metab. 22:266-274, 2011 ).
  • Diagnostic markers that aid in predicting the susceptibility of a subject to develop pancreatic ⁇ -cell dysfunction and death will be helpful for treating or delaying the progression of pancreatic ⁇ -cell disorders (e.g., type 1 or type 2 diabetes) in subjects.
  • Patent application WO 2010/072383 discloses ARMET (synonym of MANF) as a marker of pancreatic cancer and its use in a method of diagnostic.
  • the invention is based, in part, on the discovery that stressed pancreatic ⁇ -cells produce and secrete soluble mesencephalic astrocyte-derived neurotrophic factor (MANF), soluble MANF protects pancreatic ⁇ -cells from endoplasmic reticulum stress-induced apoptosis, and soluble MANF maintains endoplasmic reticulum redox homeostasis in pancreatic ⁇ -cells.
  • MANF mesencephalic astrocyte-derived neurotrophic factor
  • a pancreatic ⁇ -cell disorder in accordance with the claims. Also provided in the disclosure are methods of diagnosing a pancreatic ⁇ -cell disorder in a subject, predicting a subject's risk of developing a pancreatic ⁇ -cell disorder, monitoring pancreatic ⁇ -cell function or pancreatic ⁇ -cell mass in a subject (e.g., a subject at risk of developing a pancreatic ⁇ -cell disorder), identifying a subject having an increased risk of developing a pancreatic ⁇ -cell disorder, , selecting a subject for participation in a clinical study, and detecting endoplasmic reticulum stress in a pancreatic ⁇ -cell. These methods include determining at least one level of soluble MANF (e.g., endogenous levels of soluble MANF in a biological sample from the subject or in a culture medium).
  • soluble MANF e.g., endogenous levels of soluble MANF in a biological sample from the subject or in a culture medium.
  • Also provided in the disclosure are methods (e.g., in vitro methods) of diagnosing a pancreatic ⁇ -cell disorder in a subject that include determining a level of soluble mesencephalic astrocyte-derived neurotrophic factor (MANF) in a biological sample from a subject; comparing the level of soluble MANF in the biological sample to a reference level of soluble MANF; and identifying a subject having an elevated level of soluble MANF in the biological sample as compared to the reference level as having a pancreatic ⁇ -cell disorder.
  • MANF soluble mesencephalic astrocyte-derived neurotrophic factor
  • Also provided in the disclosure are methods (e.g., in vitro methods) of predicting a subject's risk of developing a pancreatic ⁇ -cell disorder that include: determining a level of soluble mesencephalic astrocyte-derived neurotrophic factor (MANF) in a biological sample from a subject; comparing the level of soluble MANF in the biological sample to a reference level of soluble MANF; and identifying a subject having an elevated level of soluble MANF in the biological sample compared to the reference level as having an increased risk of developing a pancreatic ⁇ -cell disorder, or identifying a subject that has a decrease or no significant difference in the level of soluble MANF in the biological sample as compared to the reference level as having a normal or decreased risk of developing a pancreatic ⁇ -cell disorder.
  • MANF soluble mesencephalic astrocyte-derived neurotrophic factor
  • Also provided in the disclosure are methods (e.g., in vitro methods) of monitoring pancreatic ⁇ -cell function or pancreatic ⁇ -cell mass in a subject that include: determining a level of soluble mesencephalic astrocyte-derived neurotrophic factor (MANF) in a biological sample from the subject at a first time point; determining a level of soluble MANF in a biological sample from the subject at a second time point; comparing the level of soluble MANF in the biological sample at the second time point to the level of soluble MANF in the biological sample at the first time point; and identifying a subject having an elevated level of soluble MANF in the biological sample at the second time point compared to the level of soluble MANF in the biological sample at the first time point as having a decrease in pancreatic ⁇ -cell function or a decrease in pancreatic ⁇ -cell, or identifying a subject having a decrease or no significant change in the level of soluble MANF in the biological sample at the second time point
  • Also provided in the disclosure are methods of treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject, methods (e.g., in vitro methods) of reducing endoplasmic reticulum stress in a pancreatic ⁇ -cell, or methods (e.g., in vitro methods) of reducing or delaying endoplasmic reticulum stress-induced apoptotic cell death in a population of two or more pancreatic ⁇ -cells. These methods include the administration of an effective amount of a soluble MANF or apomorphine to a subject, or contacting the pancreatic ⁇ -cell or the population of pancreatic ⁇ -cells with a soluble MANF or apomorphine.
  • the soluble MANF contains a sequence that is at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a mammalian soluble MANF protein sequence (e.g., any one of SEQ ID NOS: 2 and 4-7).
  • a mammalian soluble MANF protein sequence e.g., any one of SEQ ID NOS: 2 and 4-7.
  • the method includes administering apomorpine, the subject does not have erectile dysfunction or Parkinson's disease.
  • soluble MANF comprising a sequence that is at least 80% identical to SEQ ID NO: 2
  • apomorphine in the manufacture of a medicament for treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject.
  • isolated soluble MANF e.g., an isolated soluble MANF comprising comprising a sequence that is at least 80% identical to SEQ ID NO: 2 for use in treating or delaying the onset of a pancreatic ⁇ -cell disorder in a in accordance with the claims.
  • Also provided in the disclosure are methods (e.g., in vitro methods) of screening for a candidate compound useful for treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject, methods (e.g., in vitro methods) for decreasing endoplasmic reticulum stress in a pancreatic ⁇ -cell, and methods (e.g., in vitro methods) for reducing or delaying endoplasmic reticulum stress-induced apoptotic cell death in pancreatic ⁇ -cells.
  • These methods include providing a pancreatic ⁇ -cell, contacting the pancreatic ⁇ -cell with a candidate compound, determining the level of soluble MANF produced by the pancreatic ⁇ -cell in the presence of the candidate compound, comparing the level of soluble MANF produced by the pancreatic ⁇ -cell to a reference level of soluble MANF, and selecting a compound that is associated with an elevated level of soluble MANF being produced by the pancreatic ⁇ -cell compared to the reference level as a candidate compound for treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject.
  • an elevated level of soluble MANF produced by the pancreatic ⁇ -cell compared to the reference level indicates that the candidate compound may be useful for treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject, decreasing endoplasmic reticulum stress in a pancreatic ⁇ -cell, or reducing or delaying endoplasmic reticulum stress-induced apoptotic cell death in pancreatic ⁇ -cells.
  • a mammalian cell e.g., a pancreatic ⁇ -cell
  • a reporter protein containing a BiP signal sequence e.g., a redox-sensitive fluorescent protein
  • a redox-sensitive fluorescent protein e.g., a redox-sensitive green fluorescent protein
  • the amino acid sequence of KDEL e.g., KDEL
  • contacting the cell with a test compound determining the amount of oxidized reporter protein present in the cell
  • the reference level is the amount of oxidized reporter protein present in a mammalian cell in the absence of the candidate agent.
  • the reference level is a threshold level of oxidized reporter protein.
  • a mammalian cell e.g., a pancreatic ⁇ -cell
  • a reporter protein containing a BiP signal sequence, a redox-sensitive fluorescent protein (e.g., a green fluorescent protein), and the amino acid sequence of KDEL e.g., a test compound
  • determining the amount of reduced reporter protein present in the cell e.g., a redox-sensitive fluorescent protein
  • a reference level is the amount of reduced reporter protein present in a mammalian cell in the absence of the candidate agent.
  • the reference level is a threshold level of reduced reporter protein.
  • kits including an antibody or antigen-binding antibody fragment that binds specifically to a soluble MANF (e.g., a human soluble MANF), and at least one antibody or antigen-binding antibody fragment that binds to a protein selected from insulin, C-protein, and islet amyloid polypeptide (IAPP).
  • a soluble MANF e.g., a human soluble MANF
  • IAPP islet amyloid polypeptide
  • compositions that contain a soluble MANF protein (e.g., a soluble MANF protein containing a sequence at least 80% identical to SEQ ID NO: 2) and/or apomorphine, and at least one additional agent for treating a pancreatic ⁇ -cell disorder (e.g., pioglitazone, TUDCA, GLP-1, or a DPP-4 inhibitor (e.g., sitagliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin, and alogliptin)).
  • a pancreatic ⁇ -cell disorder e.g., pioglitazone, TUDCA, GLP-1, or a DPP-4 inhibitor (e.g., sitagliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin, and aloglipt
  • pancreatic ⁇ -cell disorder is meant a disease that includes, as part of its pathogenesis, a decrease in pancreatic ⁇ -cell function (e.g., insulin secretion) or a decrease in the number of viable insulin-secreting pancreatic ⁇ -cells present in a subject (pancreatic ⁇ -cell mass).
  • a pancreatic ⁇ -cell disorder can be further characterized by an increase in the endoplasmic reticulum stress in a population of pancreatic ⁇ -cells (e.g., two or more pancreatic ⁇ -cells) in the subject.
  • pancreatic ⁇ -cell function As described herein a decrease in pancreatic ⁇ -cell function, a decrease in the number of viable pancreatic ⁇ -cells (pancreatic ⁇ -cell mass), or an increase in the endoplasmic reticulum stress in pancreatic ⁇ -cells present in a subject can be detected indirectly using the methods described herein or other methods known in the art.
  • pancreatic ⁇ -cell disorders include type 1 diabetes (diabetes mellitus), type 2 diabetes, and Wolfram syndrome.
  • pancreatic ⁇ -cell function is meant a biological activity that is used to describe a mammalian (e.g., human) pancreatic ⁇ -cell (e.g., an activity that is specifically unique to a pancreatic ⁇ -cell).
  • pancreatic ⁇ -cell function include the synthesis and secretion of insulin, the synthesis and secretion of islet amyloid polypeptide (IAPP), and the synthesis and section of C-peptide.
  • IAPP islet amyloid polypeptide
  • Methods for detecting the synthesis and secretion of insulin, IAPP, and C-peptide are known in the art.
  • Pancreatic ⁇ -cell function can also be detected indirectly using the methods described herein, as well as methods known in the art (e.g., determining blood glucose levels and determining glycated hemoglobin A1C levels).
  • pancreatic ⁇ -cell mass is meant the total number of viable insulin-secreting pancreatic ⁇ -cells in a mammal (e.g., a human).
  • Methods for indirectly determining the pancreatic ⁇ -cell mass in a subject are described herein. Additional methods for indirectly determining the pancreatic ⁇ -cell mass in a subject are known in the art (e.g., determining blood glucose levels and determining glycated hemoglobin A1C levels).
  • the pancreatic ⁇ -cell mass may represent the total number of endogenous viable pancreatic ⁇ -cells in a subject or may represent the sum of the number of endogenous viable pancreatic ⁇ -cells in a subject plus the number of viable pancreatic ⁇ -cells transplanted into the subject (e.g., autograft, homograft, or xenografted viable pancreatic ⁇ -cells).
  • soluble MANF is meant a protein containing a sequence that is at least 80% identical to a sequence of a soluble mammalian form of mesencephalic astrocyte-derived neutrophic factor (MANF).
  • a soluble MANF can be protein containing a sequence that is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of SEQ ID NOS: 2 and 4-7, i.e., to the full length of SEQ ID NOs: 2 or 4-7.
  • a soluble MANF can be a wildtype mammalian soluble MANF (e.g., SEQ ID NO: 2 and 4-7).
  • a soluble MANF protein can be administered as a therapeutic treatment (e.g., as a recombinant or purified endogenous protein using any of the methods described herein).
  • a purified soluble MANF protein can be, e.g., at least 80% (e.g., at least 85%, 90%, 95%, or 99%) pure by dry weight. Additional modified forms of soluble MANF are described herein.
  • increase or “elevated” is meant an observable, detectable, or significant increase in a level as compared to a reference level or a level measured at an earlier or later time point in the same subject (e.g., in a biological sample from the same subject).
  • decrease is meant an observable, detectable, or significant decrease in a level as compared to a reference level or a level measured at an earlier or later time point in the same subject (e.g., in a biological sample from the same subject).
  • a compound that is associated with an elevated level of soluble MANF is meant a compound that induces or results in an elevated level of soluble MANF (e.g., protein or mRNA) present, produced by, or secreted by a mammalian cell that is contacted with the compound, as compared to the level of soluble MANF (e.g., protein or mRNA) present, produced by, or secreted by a control mammalian cell (e.g., the same or the same type of mammalian cell) in the absence of the compound.
  • soluble MANF e.g., protein or mRNA
  • risk of developing disease is meant the relative probability that a subject will develop a pancreatic ⁇ -cell disorder in the future as compared to a control subject or population (e.g., a healthy subject or population, or a subject or population with no family history of a pancreatic ⁇ -cell disorder).
  • a control subject or population e.g., a healthy subject or population, or a subject or population with no family history of a pancreatic ⁇ -cell disorder.
  • methods for determining a subject's risk of developing a pancreatic ⁇ -cell disorder (in the future) that include determining a level of soluble MANF.
  • treating includes reducing the number of symptoms or reducing the severity, duration, or frequency of one or more symptoms of disease (e.g., a pancreatic ⁇ -cell disorder) in a subject.
  • treating can also include reducing the risk of developing a pancreatic ⁇ -cell disorder in a subject (in the future) or delaying the onset of one or more symptoms of a pancreatic ⁇ -cell disorder in a subject.
  • a pancreatic ⁇ -cell disorder By the phrase “delaying the onset of a pancreatic ⁇ -cell disorder” is meant an increase in the length of time before one or more symptoms of a pancreatic ⁇ -cell disorder are observed in a subject.
  • the subject can be previously identified as having an increased risk of developing a pancreatic ⁇ -cell disorder.
  • a subject can be identified as having an increased risk of developing a pancreatic ⁇ -cell disorder using the methods described herein or by the observation of a family history of a pancreatic ⁇ -cell disorder (e.g., type 2 diabetes).
  • biological sample includes any sample collected from a subject that includes a biological fluid.
  • the biological sample can include blood, serum, plasma, urine, cerebrospinal fluid, saliva, bile, gastric juice, or breast milk.
  • endoplasmic reticulum stress is meant an imbalance the endoplasmic reticulum between the production of reactive oxygen species (pro-oxidant species) and the cell's or cellular organelle's ability to detoxify (remove) the reactive oxygen species (or their intermediates) that results in a shift in the redox potential of the lumen of the endoplasmic reticulum and/or an accumulation of misfolded or unfolded proteins within the lumen of the endoplasmic reticulum.
  • Endoplasmic reticulum stress triggers a unique stress pathway termed the unfolded protein response (UPR) (further described herein).
  • UTR unfolded protein response
  • Endoplasmic reticulum stress in a pancreatic ⁇ -cell can be caused by a number of molecular events (e.g., an increased level of free fatty acids in the endoplasmic reticulum, hyperinsulemia, hyper-production of VEGF, hypoxia, glucose deprivation, mutant islet amyloid polypeptide, mutant insulin, increased levels of IL-1, increased levels of IFN-K, or virus infection).
  • a variety of different chemical agents can also be used to induce endoplasmic reticulum stress (e.g., thapsigargin or tunicamycin).
  • Endoplasmic reticulum stress has been shown to shift the endoplasmic reticulum from an oxidizing environment towards a more reducing environment.
  • agents that have the ability to shift the endoplasmic reticulum from a reducing toward an oxidizing environment under ER stress conditions will help reduce ER stress and/or may reduce or prevent ER stress-induced apoptotic cell death.
  • Endoplasmic reticulum stress can be detected using a variety of different methods known in the art. Exemplary methods for detecting, reducing, or delaying the onset of endoplasmic reticulum stress in pancreatic ⁇ -cells are described herein.
  • pancreatic ⁇ -cells two or more pancreatic ⁇ -cells.
  • a population of pancreatic ⁇ -cells may be present in a mammalian (e.g., human) subject (e.g., a subject's endogenous pancreatic ⁇ -cells, an autograft, homograft, or xenograft of pancreatic ⁇ -cells).
  • a population of pancreatic ⁇ -cells can be cultured in vitro (tissue culture).
  • a population of pancreatic ⁇ -cells is pancreatic ⁇ -cell line (e.g., those pancreatic ⁇ -cell lines described herein).
  • a pancreatic ⁇ -cell can be derived from any mammalian species (e.g., human, monkey (e.g., chimpanzee), mouse, pig, rat, or ape).
  • a pancreatic ⁇ -cell population can be a primary cell line or an immortalized cell line.
  • pancreatic ⁇ -cell an insulin-producing cell that is normally present in the pancreas of a mammal in the islet of Langerhans.
  • pancreatic ⁇ -cell encompasses a pancreatic ⁇ -cell present in the body of a mammal (e.g., endogenous pancreatic ⁇ -cells, or autograft, homograft, or xenograft pancreatic ⁇ -cells) or a pancreatic ⁇ -cell cultured in vitro (e.g., an ex vivo (e.g., primary) culture of pancreatic ⁇ -cells from any mammalian species described herein or a pancreatic ⁇ -cell line (e.g., a primary or immortalized cell line).
  • a mammal e.g., endogenous pancreatic ⁇ -cells, or autograft, homograft, or xenograft pancreatic ⁇ -cells
  • the pancreatic ⁇ -cell present in a mammal is present in the pancreas. In some embodiments, the pancreatic ⁇ -cell present in a mammal is located in a tissue other than the pancreas (e.g., in liver tissue). In other embodiments, the pancreatic ⁇ -cell is encapsulated in a device (e.g., a biocompatible polymer) that is implanted in the subject.
  • the term pancreatic ⁇ -cell also encompasses a pancreatic ⁇ -cell in a mammalian (e.g., human, pig, rat, and mouse) cell line or a primary mammalian (e.g., human, pig, rat, and mouse) cell culture.
  • the pancreatic ⁇ -cell can be genetically manipulated using molecular biology techniques to express one or more recombinant proteins (e.g., an insulin) and/or decrease the expression of one or more endogenous proteins.
  • endoplasmic reticulum-induced apoptotic cell death programmed cell death that is triggered by stress in the endoplasmic reticulum of a cell (e.g., a pancreatic ⁇ -cell).
  • the endoplasmic reticulum stress that induces apoptotic cell death induces the unfolded protein response (UPR) pathway in the cell (e.g., a pancreatic ⁇ -cell).
  • UPR unfolded protein response
  • endoplasmic reticulum stress can be caused by a number of agents (e.g., biological and chemical agents).
  • determining a level is meant the use of one or more scientific techniques (e.g., molecular biology, molecular genetics, immunological, and biochemical methods or assays) to assess the level of a particular molecule (e.g., in a biological sample or cell culture medium).
  • the phrase determining a level includes the physical contacting of one or more reagents that specifically bind to a particular molecule (e.g., an antibody or antigen-binding fragment of an antibody) to a sample (e.g., a biological sample or cell culture medium).
  • second time point generally means any point in time that occurs after a first time point (e.g., time of admission).
  • a second time point can occur, e.g., at least 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 6 months, 1 year, or 2 years after the first time point.
  • a subject can be administered a treatment between the first time point and the second time point.
  • redox-sensitive fluorescent protein is a protein that changes its fluorescence properties (e.g., change in excitation and/or emission spectrum (e.g., peak excitation or emission wavelengths) upon a change in its redox environment (e.g., a change in the redox environment of the endoplasmic reticulum). In some embodiments, this change in the fluorescence properties of the protein can occur, e.g., as a result of the formation and/or breakage of one or more disulfide bonds.
  • redox-sensitive fluorescent proteins include redox-oxidation sensitive green fluorescent protein (roGFP) and redox-sensitive yellow fluorescent protein (rxYFP). Additional redox-sensitive fluorescent proteins are known in the art.
  • the invention is based, at least in part, on the discovery that soluble MANF is secreted by stressed pancreatic ⁇ -cells, and that soluble MANF delays endoplasmic reticulum stress-induced pancreatic ⁇ -cell apoptotic cell death and reduces fluctuation in the redox state of the endoplasmic reticulum in pancreatic ⁇ -cells exposed to agents or conditions that induce endoplasmic reticulum stress.
  • the disclosure provides methods for diagnosing a pancreatic ⁇ -cell disorder, predicting a subject's risk of developing a pancreatic ⁇ -cell disorder, monitoring pancreatic ⁇ -cell function and pancreatic ⁇ -cell mass in a subject (e.g., a subject at risk of developing a pancreatic ⁇ -cell disorder), monitoring the efficacy of treatment of a pancreatic ⁇ -cell disorder in a subject, identifying a subject having an increased risk of developing a pancreatic ⁇ -cell disorder, selecting a subject for treatment of pancreatic ⁇ -cell disorder, selecting a subject for participation in a clinical study, and detecting endoplasmic reticulum stress in a pancreatic ⁇ -cell are provided. These methods include determining at least one level of soluble MANF (e.g., in a biological sample from the subject or in a culture medium).
  • soluble MANF comprising a sequence that is at least 80% identical to SEQ ID NO: 2 and has a biological activity of soluble MANF in methods of treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject, which reduce endoplasmic reticulum stress in a pancreatic ⁇ -cell, and reduce or delay endoplasmic reticulum stress-induced apoptotic cell death in a population of two or more pancreatic ⁇ -cells are also provided. These uses include the administration of an effective amount of a soluble MANF to a subject, or contacting a pancreatic ⁇ -cell or a population of pancreatic ⁇ -cells with a soluble MANF. In some embodiments of the disclosure, e.g., wherein the method includes administering apomorpine, the subject does not have erectile dysfunction or Parkinson's disease.
  • these methods include providing a pancreatic ⁇ -cell, contacting the pancreatic ⁇ -cell with a candidate compound, determining the level of soluble MANF produced by the pancreatic ⁇ -cell in the presence of the candidate compound, and comparing the level of soluble MANF produced by the pancreatic ⁇ -cell to a reference level of soluble MANF.
  • these methods include providing a mammalian cell (e.g., a pancreatic ⁇ -cell) expressing a reporter protein containing a BiP signal sequence, a redox-sensitive fluorescent protein (e.g., a redox-sensitive green fluorescent protein or redox-sensitive yellow fluorescent protein), and the amino acid sequence of KDEL; contacting the cell with a test compound; determining the amount of oxidized reporter protein present in the cell; and comparing the amount of oxidized reporter protein present in the cell to a reference level; where an elevated level of oxidized reporter protein in the cell compared to the reference level indicates that the candidate compound may be useful for treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject, decreasing endoplasmic reticulum stress in a pancreatic ⁇ -cell, and/or reducing or delaying endoplasmic reticulum stress-induced apoptotic cell death in pancreatic ⁇ -cells.
  • a mammalian cell e.g
  • these methods include providing a mammalian cell (e.g., a pancreatic ⁇ -cell) expressing a reporter protein containing a BiP signal sequence, a redox-sensitive fluorescent protein (e.g., a redox-sensitive green fluorescent protein or redox-sensitive yellow fluorescent protein), and the amino acid sequence of KDEL; contacting the cell with a test compound; determining the amount of reduced reporter protein present in the cell; and comparing the amount of reduced reporter protein present in the cell to a reference level; where a decreased level of reduced reporter protein in the cell compared to the reference level indicates that the candidate compound may be useful for treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject, decreasing endoplasmic reticulum stress in a pancreatic ⁇ -cell, and/or reducing or delaying endoplasmic reticulum stress-induced apoptotic cell death in pancreatic ⁇ -cells.
  • a mammalian cell e.g., a pan
  • Pancreatic ⁇ -cell disorders are a class of diseases that are characterized by a progressive decrease in pancreatic ⁇ -cell dysfunction or pancreatic ⁇ -cell mass in the subject.
  • Pancreatic ⁇ -cell functions that can be decreased in a subject having a pancreatic ⁇ -cell disorder include, without limitation, insulin production and secretion, C-peptide production and secretion, and islet amyloid polypeptide (IAPP) production and secretion.
  • IAPP islet amyloid polypeptide
  • pancreatic ⁇ -cell disorders include type 1 diabetes (diabetes mellitus), type 2 diabetes, and Wolfram syndrome.
  • Pancreatic ⁇ -cell disorders can occur in humans at any age, e.g., in infants, children, adults, and elderly subjects.
  • a pancreatic ⁇ -cell disorder can occur in a subject having an age of greater than 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90.
  • a health care professional may diagnose a subject as having a pancreatic ⁇ -cell disorder by the assessment of one or more (e.g., two, three, four, or five) symptoms of a pancreatic ⁇ -cell disorder in the subject.
  • Non-limiting symptoms of a pancreatic ⁇ -cell disorder in a subject include a significant increase in blood glucose levels (e.g., fasting blood glucose levels) compared to a healthy individual or population (e.g., a fasting blood glucose level of greater than 100 mg/dL or greater than 120 mg/dL), a significant increase in glycated hemoglobin levels (hemoglobin A1C level) compared to a healthy individual or population (e.g., a hemoglobin A1C level greater than 6.5%, greater than 7.0%, or greater than 8.0%), increased thirst, frequent urination, extreme hunger, unexplained weight loss, presence of ketones in the urine, fatigue, blurred vision, slow-healing sores, mild high blood pressure, and frequent infections.
  • blood glucose levels e.g., fasting blood glucose levels
  • hemoglobin A1C level glycated hemoglobin levels
  • a healthy individual or population e.g., a hemoglobin A1C
  • a health care professional may also base a diagnosis, in part, on the subject's family history of a pancreatic ⁇ -cell disorder.
  • a health care professional may diagnose a subject as having a pancreatic ⁇ -cell disorder upon presentation of a subject to a health care facility (e.g., a clinic or a hospital).
  • a health care professional may diagnose a subject as having a pancreatic ⁇ -cell disorder while the subject is admitted in an assisted care facility.
  • a physician diagnoses a pancreatic ⁇ -cell disorder in a subject after the observation or detection of one or more symptoms in the subject.
  • a health care professional may also identify a subject as having an increased risk of developing a pancreatic ⁇ -cell disorder based on one of more of the following factors: increased weight (e.g., a body mass index of > 25 or > 30), inactivity, family history of a pancreatic ⁇ -cell disorder, race, age, diagnosis with polycystic ovary syndrome, high blood pressure, decreased high-density lipoprotein levels (e.g., below 35 mg/dL), and high levels of triglycerides (e.g., above 250 mg/dL).
  • increased weight e.g., a body mass index of > 25 or > 30
  • inactivity e.g., family history of a pancreatic ⁇ -cell disorder
  • race e.g., age, diagnosis with polycystic ovary syndrome
  • high blood pressure e.g., decreased high-density lipoprotein levels (e.g., below 35 mg/dL)
  • high levels of triglycerides
  • a pancreatic ⁇ -cell disorder in a subject e.g., a subject presenting with one or more symptoms of a pancreatic ⁇ -cell disorder or a subject not presenting with a symptom of a pancreatic ⁇ -cell disorder (e.g., an undiagnosed and/or asymptomatic subject).
  • additional methods of identifying a subject having an increased risk of developing a pancreatic ⁇ -cell disorder are also provided herein.
  • MEF Mesencephalic Astrocyte-Derived Neurotrophic Factor
  • An endogenous level of soluble MANF protein, as described herein, can be detected in any of the methods described herein, e.g., as a marker for diagnosing a pancreatic ⁇ -cell disorder, predicting a subject's risk of developing a pancreatic ⁇ -cell disorder, monitoring pancreatic ⁇ -cell function and pancreatic ⁇ -cell mass in a subject (e.g., a subject at risk of developing a pancreatic ⁇ -cell disorder), identifying a subject having an increased risk of developing a pancreatic ⁇ -cell disorder, selecting a subject for treatment of pancreatic ⁇ -cell disorder, selecting a subject for participation in a clinical study, and detecting endoplasmic reticulum stress in a pancreatic ⁇ -cell.
  • a marker for diagnosing a pancreatic ⁇ -cell disorder e.g., a subject's risk of developing a pancreatic ⁇ -cell disorder, monitoring pancreatic ⁇ -cell function and pancreatic ⁇ -cell
  • a purified, isolated, and/or recombinant soluble MANF protein can also be used in methods of treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject, reducing endoplasmic reticulum stress in a pancreatic ⁇ -cell, and reducing or delaying endoplasmic reticulum stress-induced apoptotic cell death in a population of two or more pancreatic ⁇ -cells.
  • MANF protein is translated as a precursor protein that is subsequently cleaved and released as a soluble protein from a cell (e.g., a pancreatic ⁇ -cell).
  • a cell e.g., a pancreatic ⁇ -cell.
  • the full-length (precursor) human MANF protein and the human soluble MANF protein are shown below.
  • the 25-amino acid signal sequence in the precursor human MANF protein is underlined below. Also shown below is the mRNA encoding the human precursor MANF protein.
  • Cow, rat, mouse, pig, fly, and zebrafish soluble MANF protein sequences have also been described, and are shown below.
  • Cow soluble MANF (SEQ ID NO: 4)
  • Rat soluble MANF (SEQ ID NO: 5)
  • Drosophila melanogaster soluble MANF (SEQ ID NO: 8)
  • Mammalian soluble MANF proteins are highly conserved, with human and cow soluble MANF proteins having 96% identity, human and mouse soluble MANF proteins having 99% identity, and human and pig soluble MANF proteins having 97% identity. Human soluble MANF protein also shares 72% identity and 88% similarity with zebrafish soluble MANF protein.
  • kits in the disclosure are methods of diagnosing a pancreatic ⁇ -cell disorder in a subject. These methods include determining (assaying) a level of soluble MANF in a biological sample from a subject, and comparing the level of soluble MANF in the biological sample to a reference level of soluble MANF. In these methods, an elevated level of soluble MANF in the biological sample as compared to the reference level of soluble MANF indicates that the subject has a pancreatic ⁇ -cell disorder, and a decrease or no significant change in the level of soluble MANF in the biological sample as compared to the reference level of soluble MANF indicates that the subject does not have a pancreatic ⁇ -cell disorder.
  • a "reference level of soluble MANF” can be a threshold level of soluble MANF, a level of soluble MANF present in a control subject or population (e.g., a subject or population that is not diagnosed as having a disease (a healthy subject or population), does not have one or more symptoms of a pancreatic ⁇ -cell disorder, and/or does not have a family history of a pancreatic ⁇ -cell disorder), or a level of soluble MANF in the same subject at an earlier time point.
  • the levels of soluble MANF can be determined using methods known in the art. For example, the levels of soluble MANF can be detected using a number of techniques known in the art that utilize antibodies that specifically bind to a soluble MANF (e.g., enzyme-linked immunosorbent assay). A number of antibodies that specifically bind to human soluble MANF are commercially available (e.g., rabbit anti-human soluble MANF antibodies available from Sigma-Aldrich and Proteintech).
  • any of the methods described in the disclosure may further include obtaining or collecting a sample from a subject (e.g., a biological sample containing a biological fluid, e.g., urine, blood, plasma, serum, or cerebrospinal fluid).
  • a biological sample containing a biological fluid, e.g., urine, blood, plasma, serum, or cerebrospinal fluid.
  • the biological sample can be stored for a period of time (e.g., at least one hour or at least 24 hours) before a level of soluble MANF is determined (e.g., storage at or below 10 °C).
  • any of the methods described in the disclosure can be performed on patients presenting to a health care facility (e.g., a hospital, clinic, or an assisted care facility).
  • the subjects may present with one or more symptoms of a pancreatic ⁇ -cell disorder (e.g., any of the symptoms of a pancreatic ⁇ -cell disorder described herein).
  • the subject may be suspected of having a pancreatic ⁇ -cell disorder.
  • the subject can also present with no symptoms (an asymptomatic subject) or just one symptom of a pancreatic ⁇ -cell disorder.
  • the subject can have a family history of a pancreatic ⁇ -cell disorder (e.g., type 2 diabetes).
  • the subject can also have an increased risk of developing a pancreatic ⁇ -cell disorder.
  • the subject can be an infant, a child, a teenager, an adult, or an elderly person.
  • the methods are performed on a subject that has a detectable or observable pancreatic ⁇ -cell mass and/or has detectable or observable amount pancreatic ⁇ -cell function (e.g., a subject that does not have a complete loss of pancreatic ⁇ -cell mass or pancreatic ⁇ -cell function).
  • a subject that does not have a complete loss of pancreatic ⁇ -cell mass or pancreatic ⁇ -cell function e.g., a subject that does not have a complete loss of pancreatic ⁇ -cell mass or pancreatic ⁇ -cell function.
  • Methods of detecting pancreatic ⁇ -cell function are described herein.
  • Pancreatic ⁇ -cell mass can be detected indirectly by observing pancreatic ⁇ -cell function in a subject or using methods known in the art (e.g., the methods described in U.S. Patent Application Publication No. 20110123443 ).
  • the diagnostic methods described in the disclosure can be performed by any health care professional (e.g., a physician, a laboratory technician, a nurse, a physician's assistant, and a nurse's assistant).
  • the diagnostic methods described herein can be used in combination with one or more additional diagnostic testing methods known in the art or described herein (e.g., the observation or assessment of one or more symptoms of a pancreatic ⁇ -cell disorder in a subject, e.g., blood glucose monitoring, glycated hemoglobin analysis, level of insulin, level of IAPP, level of C-peptide, or ketones in the urine).
  • the diagnostic methods described in the disclosure can be performed on a subject identified as having an increased risk of developing a pancreatic ⁇ -cell disorder (e.g., a subject identified as having an increased risk of developing a pancreatic ⁇ -cell disorder using any of the methods described herein).
  • the diagnostic methods described herein can be performed periodically (e.g., at least once every month, two months, six months, or year) for a subject that has been identified as having an increased risk of developing a pancreatic ⁇ -cell disorder.
  • Some embodiments of the disclosure further include collecting the biological sample from the subject.
  • Some embodiments of the disclosure further include administering to the subject identified as having, or at risk of developing, a pancreatic ⁇ -cell disorder a treatment for a pancreatic ⁇ -cell disorder (e.g., an isolated, purified, or recombinant soluble MANF protein, pioglitazone, GLP-1, or a DPP-4 inhibitor (e.g., sitagliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin, and alogliptin), or any of the compositions described herein, e.g., an therapeutically effective dose of a soluble MANF protein containing a sequence at least 90% identical to SEQ ID NO: 2 and/or apomorphine).
  • a treatment for a pancreatic ⁇ -cell disorder e.g., an isolated, purified, or recombinant soluble MANF protein, pioglitazone, GLP-1
  • Some embodiments of the disclosure further include performing additional tests to confirm the diagnosis of a pancreatic ⁇ -cell disorder in the subject.
  • Some embodiments of the disclosure include selecting the subject for periodic glucose monitoring (e.g., periodic self-glucose monitoring using a glucometer) or any of the monitoring methods described herein.
  • Some embodiments of the disclosure further include selecting the subject for periodic medical evaluation by a physician or a health care professional (e.g., periodic visits of at least once every year, at least once every six months, at least once every three months, at least once every two months, or at least once a month).
  • Some embodiments of the disclosure further include recording the results of the diagnostic test in the subject's medical records, or performing a diagnostic test for a pancreatic ⁇ -cell disorder on one or more lineal family members of a subject diagnosed as having a pancreatic ⁇ -cell disorder using the methods described herein.
  • the levels of soluble MANF can be determined using methods known in the art.
  • the levels of soluble MANF can be detected using a number of techniques known in the art that utilize antibodies that specifically bind to soluble MANF (e.g., enzyme-linked immunosorbent assay).
  • the methods further include obtaining or collecting a sample from a subject (e.g., a biological sample containing a biological fluid, e.g., murine, blood, plasma, serum, or cerebrospinal fluid).
  • any of the methods described in the disclosure can be performed on patients presenting to a health care facility (e.g., a hospital, clinic, or an assisted care facility).
  • the methods are performed on a subject as part of a periodic physical examination by a health care professional.
  • the subjects may present with one or more symptoms of a pancreatic ⁇ -cell disorder (e.g., any of the symptoms of a pancreatic ⁇ -cell disorder described herein).
  • the subject may be suspected of having a pancreatic ⁇ -cell disorder.
  • the subject can also present with no symptoms (an asymptomatic subject) or just one symptom of a pancreatic ⁇ -cell disorder.
  • the subject can have a family history of a pancreatic ⁇ -cell disorder (e.g., type 2 diabetes).
  • the subject can be an infant, a child, a teenager, an adult, or an elderly person.
  • a subject at risk of developing a pancreatic ⁇ -cell disorder can be assessed using any of the methods described herein (see, e.g., the next section).
  • any health care professional e.g., a physician, a laboratory technician, a nurse, a physician's assistant, and a nurse's assistant.
  • These methods can be used in combination with any additional methods known in the art for identifying a subject at risk of developing a pancreatic ⁇ -cell disorder (e.g., assessment of one of more of the following factors: increased weight, inactivity, family history of a pancreatic ⁇ -cell disorder, race, age, diagnosis with polycystic ovary syndrome, high blood pressure, decreased high-density lipoprotein levels (e.g., below 35 mg/dL), and high levels of triglycerides (e.g., above 250 mg/dL)).
  • a subject identified as having an increased risk of developing a pancreatic ⁇ -cell disorder can be monitored using any of the methods described herein
  • Some embodiments of the disclosure further include administering to a subject identified as having an increased risk of developing a pancreatic ⁇ -cell disorder a treatment for a pancreatic ⁇ -cell disorder (e.g., an isolated, purified, or recombinant soluble MANF protein, pioglitazone, GLP-1, or a DPP-4 inhibitor (e.g., sitagliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin, and alogliptin), or any of the compositions described herein, e.g., a therapeutically effective dose of a soluble MANF protein containing a sequence at least 90% identical to SEQ ID NO: 2 and/or apomorphine).
  • a treatment for a pancreatic ⁇ -cell disorder e.g., an isolated, purified, or recombinant soluble MANF protein, pioglitazone, GLP-1
  • Some embodiments of the disclosure include selecting the subject identified as having an increased risk of developing a pancreatic ⁇ -cell disorder for periodic glucose monitoring (e.g., periodic self-glucose monitoring using a glucometer). Some embodiments of the disclosure further include selecting a subject identified as having an increased risk of developing a pancreatic ⁇ -cell disorder for periodic medical evaluation by a physician or a health care professional (e.g., periodic visits of at least once every year, at least once every six months, at least once every three months, at least once every two months, or at least once a month).
  • Some embodiments of the disclosure further include recording the results of the test in the subject's medical records, or performing a similar test or any art-known test to determine the risk of developing a pancreatic ⁇ -cell disorder in one or more lineal family members of a subject identified as having an increased risk of developing a pancreatic ⁇ -cell disorder using any of the methods described herein.
  • pancreatic ⁇ -cell dysfunction in a subject e.g., a subject at risk of developing a pancreatic ⁇ -cell disorder, a subject having a pancreatic ⁇ -cell disorder, or a subject that has received a pancreatic ⁇ -cell transplant.
  • methods include determining (assaying) a level of soluble MANF in a biological sample from the subject at a first time point, determining (assaying) a level of soluble MANF in a biological sample from the subject at a second time point, and comparing the level of soluble MANF in the biological sample at the second time point to the level of soluble MANF in the biological sample at the first time point.
  • An elevated level of soluble MANF in the biological sample at the second time point compared to the level of soluble MANF at the first time point indicates a decrease (e.g., a significant, observable, or detectable decrease) in pancreatic ⁇ -cell function or a decrease in pancreatic ⁇ -cell mass in the subject.
  • a decrease or no significant change in the level of soluble MANF in the biological sample at the second time point compared to the level of soluble MANF in the biological sample at the first time point indicates no change or an increase in pancreatic ⁇ -cell function or pancreatic ⁇ -cell mass in the subject.
  • the methods are performed on a subject that has a detectable or observable pancreatic ⁇ -cell mass and/or has detectable or observable amount pancreatic ⁇ -cell function at the first and the second time point (e.g., a subject that does not have a complete loss of pancreatic ⁇ -cell mass or pancreatic ⁇ -cell function at the first and the second time point).
  • the second time point can be at least 6 hours (e.g., at least 12 hours, 18 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years) after the first time point.
  • the first time point can be the time of admittance into a medical facility or within 1 week of the first presentation of at least one symptom of a pancreatic ⁇ -cell disorder.
  • the methods can further include determining (assaying) a level of soluble MANF in a biological sample from the subject at one or more additional, later time points.
  • determining (assaying) a level of soluble MANF in a biological sample from the subject at one or more additional, later time points an elevated level of soluble MANF in a later sample (collected later in chronological time) compared to the level of soluble MANF in an earlier (e.g., immediately prior) sample (collected earlier in chronological time) indicates a decrease (e.g., a significant, observable, or detectable decrease) in pancreatic ⁇ -cell function or a decrease in pancreatic ⁇ -cell mass in the subject.
  • a decrease or no significant change in the level of soluble MANF in a later sample (collected later in chronological time) compared to the level of soluble MANF in an earlier (e.g., immediately prior) sample (collected earlier in chronological time) indicates no change or an increase in pancreatic ⁇ -cell function or pancreatic ⁇ -cell mass in the subject.
  • these methods are performed on a subject that has a detectable or observable pancreatic ⁇ -cell mass and/or has detectable or observable amount pancreatic ⁇ -cell function at the first, the second, and the one or more additional time points (e.g., a subject that does not have a complete loss of pancreatic ⁇ -cell mass or pancreatic ⁇ -cell function at the first, the second, and the one or more additional time points).
  • the subject can have previously received a pancreatic ⁇ -cell transplant, such that these methods monitor, in part, the pancreatic ⁇ -cell function and the pancreatic ⁇ -cell mass of the pancreatic ⁇ -cell transplanted into the subject.
  • the transplanted pancreatic ⁇ -cells are autografted, homografted, or xenografted pancreatic ⁇ -cells.
  • the transplanted pancreatic ⁇ -cells are present within a device, or are surrounded by or placed within a biocompatible polymer.
  • the transplanted pancreatic ⁇ -cells are present within a tissue other than the pancreas (e.g., liver tissue).
  • these methods are performed in a subject within 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year of pancreatic ⁇ -cell transplantation.
  • the first time point is shortly after (e.g., within 1 week or 2 weeks) of the transplantation procedure.
  • the levels of soluble MANF can be determined using methods known in the art. For example, the levels of soluble MANF can be detected using a number of techniques known in the art that utilize antibodies that specifically bind to soluble MANF (e.g., enzyme-linked immunosorbent assay). In some embodiments, the methods further include obtaining or collecting a sample or at least two samples from a subject (e.g., a biological sample containing a biological fluid, e.g., urine, blood, plasma, serum, or cerebrospinal fluid).
  • a biological sample containing a biological fluid e.g., urine, blood, plasma, serum, or cerebrospinal fluid.
  • any of the methods described in the disclosure can be performed on patients presenting to a health care facility (e.g., a hospital, clinic, or an assisted care facility).
  • the methods are performed on a subject as part of a periodic examination by a health care professional.
  • the subjects may present with one or more symptoms of a pancreatic ⁇ -cell disorder (e.g., any of the symptoms of a pancreatic ⁇ -cell disorder described herein).
  • the subject can also present with no symptoms (an asymptomatic subject) or just one symptom of a pancreatic ⁇ -cell disorder.
  • the subject can have a family history of a pancreatic ⁇ -cell disorder (e.g., type 2 diabetes).
  • the subject can be an infant, a child, a teenager, an adult, or an elderly person.
  • a subject identified as having a decrease (e.g., a significant, observable, or detectable decrease) in pancreatic ⁇ -cell function or a decrease in pancreatic ⁇ -cell mass can be administered a treatment for a pancreatic ⁇ -cell disorder (e.g., an isolated, purified, or recombinant soluble MANF protein, pioglitazone, GLP-1, or a DPP-4 inhibitor (e.g., sitagliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin, and alogliptin), or any of the compositions described herein, e.g., an therapeutically effective dose of a soluble MANF protein containing a sequence at least 90% identical to
  • Some embodiments further include the additional detection or assessment of one or more (e.g., two, three, or four) other markers of pancreatic ⁇ -cell dysfunction in the subject (e.g., decreased C-peptide production and secretion, decreased insulin production and secretion, decreased IAPP production and secretion, increased blood glucose levels, increased glycated hemoglobin levels, and the presence of ketones in a biological fluid of the subject (e.g., in the urine)).
  • markers of pancreatic ⁇ -cell dysfunction e.g., decreased C-peptide production and secretion, decreased insulin production and secretion, decreased IAPP production and secretion, increased blood glucose levels, increased glycated hemoglobin levels, and the presence of ketones in a biological fluid of the subject (e.g., in the urine)
  • methods for detecting one or more additional markers of a pancreatic ⁇ -cell dysfunction are known in the art.
  • Some embodiments of the disclosure further include administering to a subject identified as having an a decrease in pancreatic ⁇ -cell function or a decrease in pancreatic ⁇ -cell mass a treatment for a pancreatic ⁇ -cell disorder (e.g., an isolated, purified, or recombinant soluble MANF protein, pioglitazone, GLP-1, or a DPP-4 inhibitor (e.g., sitagliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin, and alogliptin), or any of the compositions described herein, e.g., an therapeutically effective dose of a soluble MANF protein containing a sequence at least 90% identical to SEQ ID NO: 2 and/or apomorphine).
  • a pancreatic ⁇ -cell disorder e.g., an isolated, purified, or recombinant soluble MANF protein, pioglit
  • Some embodiments of the disclosure include selecting the subject identified as having a decrease in pancreatic ⁇ -cell function or a decrease in pancreatic ⁇ -cell mass for periodic glucose monitoring (e.g., periodic self-glucose monitoring using a glucometer). Some embodiments of the disclosure further include selecting a subject identified as having a decrease in pancreatic ⁇ -cell function or a decrease in pancreatic ⁇ -cell mass for periodic medical evaluation by a physician or a health professional (e.g., periodic visits of at least once every year, at least once every six months, at least once every three months, at least once every two months, at least once a month, or at least once a week).
  • Some embodiments of the disclosure further include recording the results of the test in the subject's medical records, or performing a similar test or any art-known test to monitor pancreatic ⁇ -cell function or pancreatic ⁇ -cell mass in one or more lineal family members of a subject identified as having a decrease in pancreatic ⁇ -cell function or a decrease in pancreatic ⁇ -cell mass. Some embodiments of the disclosure further include selecting a subject having a decrease in pancreatic ⁇ -cell function or a decrease in pancreatic ⁇ -cell mass for pancreatic ⁇ -cell transplantation.
  • Also provided in the disclosure are methods of Monitoring the efficacy of treatment of a pancreatic ⁇ -cell dysfunction in a subject e.g., a subject diagnosed as having a pancreatic ⁇ -cell disorder.
  • These methods include determining (assaying) a level of soluble MANF in a biological sample from the subject at a first time point, determining (assaying) a level of soluble MANF in a biological sample from the subject at a second time point, and comparing the level of soluble MANF in the biological sample at the second time point to the level of soluble MANF in the biological sample at the first time point, where (i) the first time point is prior to treatment and the second time point is any time point following the initiation of treatment, or (ii) the first time point is following the initiation of treatment and the second time point is at a later time point during or after treatment; and a decreased level of soluble MANF in the biological sample at the second time point compared to the level of soluble MANF in
  • the treatment of a pancreatic ⁇ -cell disorder is the administration of one or more of an insulin (e.g., any of the forms of insulin described herein), pioglitazone, and TUDCA.
  • the treatment is transplantation of pancreatic ⁇ -cells into the subject (e.g., as described herein).
  • a decreased level of soluble MANF in the biological sample at the second time point compared to the level of soluble MANF at the first time point indicates efficacy of the treatment in the subject.
  • An increased level or no substantial change in the level of soluble MANF in the biological sample at the second time point compared to the level of soluble MANF at the first time point indicates that the treatment was not effective and/or that the present treatment should be terminated and/or an alternate therapy should be administered to the subject.
  • an increased level or no substantial change in the level of soluble MANF in the biological sample at the second time point compared to the level of soluble MANF at the first time point indicates that an increased dosage of the treatment should be administered to the subject or the treatment should be administered at an increased frequency and/or duration
  • the methods are performed on a subject that has a detectable or observable pancreatic ⁇ -cell mass and/or has detectable or observable amount pancreatic ⁇ -cell function at the first and the second time point (e.g., a subject that does not have a complete loss of pancreatic ⁇ -cell mass or pancreatic ⁇ -cell function at the first and the second time point).
  • the methods are performed on a subject that has been previously identified or diagnosed as having a pancreatic ⁇ -cell disorder.
  • Some embodiments of the disclosure further include selecting a subject that has a pancreatic ⁇ -cell disorder.
  • Some embodiments of the disclosure further include obtaining a sample from the subject.
  • Some embodiments of the disclosurefurther include administering one or more doses of a treatment to the subject.
  • the second time point can be at least 6 hours (e.g., at least 12 hours, 18 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years) after the first time point.
  • the first time point can be the time of admittance into a medical facility or within 1 week of the first presentation of at least one symptom of a pancreatic ⁇ -cell disorder.
  • the methods can further include determining (assaying) a level of soluble MANF in a biological sample from the subject at one or more additional, later time points.
  • determining (assaying) a level of soluble MANF in a biological sample from the subject at one or more additional, later time points a decreased level of soluble MANF in a later sample (collected later in chronological time) compared to the level of soluble MANF in an earlier (e.g., immediately prior) sample (collected earlier in chronological time) indicates efficacy of treatment in the subject.
  • an increase or no significant change in the level of soluble MANF in a later sample (collected later in chronological time) compared to the level of soluble MANF in an earlier (e.g., immediately prior) sample (collected earlier in chronological time) indicates that the treatment was not effective in the subject.
  • these methods are performed on a subject that has a detectable or observable pancreatic ⁇ -cell mass and/or has detectable or observable amount pancreatic ⁇ -cell function at the first, the second, and the one or more additional time points (e.g., a subject that does not have a complete loss of pancreatic ⁇ -cell mass or pancreatic ⁇ -cell function at the first, the second, and the one or more additional time points).
  • the subject can have previously received a pancreatic ⁇ -cell transplant, such that these methods monitor, in part, the efficacy of the pancreatic ⁇ -cell transplantation in the subject.
  • the transplanted pancreatic ⁇ -cells are autografted, homografted, or xenografted pancreatic ⁇ -cells.
  • the transplanted pancreatic ⁇ -cells are present within a device, or are surrounded by or placed within a biocompatible polymer.
  • the transplanted pancreatic ⁇ -cells are present within a tissue other than the pancreas (e.g., liver tissue).
  • these methods are performed in a subject within 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year of pancreatic ⁇ -cell transplantation.
  • the first time point is shortly after (e.g., within 1 week or 2 weeks) of the transplantation procedure.
  • the levels of soluble MANF can be determined using methods known in the art.
  • the levels of soluble MANF can be detected using a number of techniques known in the art that utilize antibodies that specifically bind to soluble MANF (e.g., enzyme-linked immunosorbent assay).
  • the methods further include obtaining or collecting a sample or at least two samples from a subject (e.g., a biological sample containing a biological fluid, e.g., urine, blood, plasma, serum, or cerebrospinal fluid).
  • any of the methods of the disclosure described herein can be performed on patients presenting to a health care facility (e.g., a hospital, clinic, or an assisted care facility).
  • the methods are performed on a subject as part of a periodic examination by a health care professional.
  • the subjects may be previously diagnosed with a pancreatic ⁇ -cell disorder and/or may present with one or more symptoms of a pancreatic ⁇ -cell disorder (e.g., any of the symptoms of a pancreatic ⁇ -cell disorder described herein).
  • the subject can be an infant, a child, a teenager, an adult, or an elderly person.
  • Some embodiments of the disclosure further include the additional detection or assessment of one or more (e.g., two, three, or four) other markers of a pancreatic ⁇ -cell disorder in the subject (e.g., where increased C-peptide production and secretion, increased insulin production and secretion, increased IAPP production and secretion, decreased blood glucose levels, decreased glycated hemoglobin levels, and the absence or no significant level of ketones in a biological fluid of the subject (e.g., in the urine) at the second time point as compared to the corresponding levels at the first time point further indicate that the treatment is effective).
  • Methods for detecting one or more additional markers of a pancreatic ⁇ -cell disorder are known in the art.
  • Some embodiments can further include assessing or determining the level of one or more additional markers of a pancreatic ⁇ -cell disorder, wherein the detection of one or more additional markers of a pancreatic ⁇ -cell disorder further indicates that the subject should be selected for treatment of a pancreatic ⁇ -cell disorder.
  • These one or more additional markers of a pancreatic ⁇ -cell disorder include a decrease in the level of C-peptide in a biological sample from the subject, a decrease in the level of IAPP in a biological sample from the subject, a decrease in the level of insulin in a biological sample from the subject, an increase one or more blood glucose level(s) in the subject, an increase in the glycated hemoglobin level in the subject, or the detection of ketones in a biological sample from the subject (e.g., in the urine).
  • Methods for detecting the levels of C-peptide, IAPP, insulin, blood glucose, glycated hemoglobin, and ketones in a biological sample from the subject are known in the art.
  • the levels of soluble MANF can be determined using methods known in the art. For example, the levels of soluble MANF can be detected using a number of techniques known in the art that utilize antibodies that specifically bind to soluble MANF (e.g., enzyme-linked immunosorbent assay).
  • the methods further include obtaining or collecting a sample from a subject (e.g., a biological sample containing a biological fluid, e.g., urine, blood, plasma, serum, or cerebrospinal fluid).
  • the methods of the disclosure can be performed by any health care professional (e.g., a physician, a nurse, a physician's assistant, a laboratory technician, or a nurse's assistant).
  • the subjects may present with one or more symptoms of a pancreatic ⁇ -cell disorder (e.g., any of the symptoms of a pancreatic ⁇ -cell disorder described herein).
  • the subject can also present with no symptoms or just one symptom of a pancreatic ⁇ -cell disorder.
  • the subject can be suspected of having a pancreatic ⁇ -cell disorder or the subject can have an increased risk of developing a pancreatic ⁇ -cell disorder.
  • the subject can have a family history of a pancreatic ⁇ -cell disorder (e.g., type 2 diabetes).
  • the subject can be previously diagnosed as having a pancreatic ⁇ -cell disorder.
  • the methods of the disclosure are performed on a subject that has a detectable or observable pancreatic ⁇ -cell mass and/or has detectable or observable amount pancreatic ⁇ -cell function (e.g., a subject that does not have a complete loss of pancreatic ⁇ -cell mass or pancreatic ⁇ -cell function).
  • treatments of a pancreatic ⁇ -cell disorder include, without limitation: a soluble MANF (e.g., a soluble MANF protein that contains a sequence at least 80% identical to SEQ ID NO: 2, i.e., to the full length of SEQ ID NO:2), apomorphine, rapid-acting insulin (e.g., aspart or lispro insulin), short-acting (e.g., regular insulin), intermediate-acting insulin (e.g., neutral protamine Hagedorn or NPH insulin), long-acting insulin (e.g., ultralente insulin), insulin glargine, insulin detemir, pramlintide, incretin mimetics (e.g., exenatide), sulfonylureas (e.g., chorpropamide, glipizide, glyburide, and glimepiride), meglitinides (e.g., re
  • Treatments of a pancreatic ⁇ -cell disorder can include one or more (e.g., two, three, or four) of the above agents used in any combination.
  • the method includes administering apomorpine, the subject does not have erectile dysfunction or Parkinson's disease.
  • Some embodiments of these methods of the disclosure further include administering to the subject at least one (e.g., at two, three, or four) treatment for a pancreatic ⁇ -cell disorder (e.g., one or more of the treatments of a pancreatic ⁇ -cell disorder described herein or known in the art).
  • some embodiments of these methods further include administering at least one (e.g., at least two, four, six, eight, or ten) dose of any of the pharmaceutical compositions described herein.
  • the treatment of a pancreatic ⁇ -cell disorder can continue over a period of time of at least 1 week, 1 month, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years.
  • the treatment can be administered periodically to the subject (e.g., once a day, twice a day, three times a day, four times a day, once a week, twice a week, three times a week, four times a week, once a month, twice a month, three times a month, or four times a month).
  • a subject having an elevated level of soluble MANF as compared to the reference level is selected for periodic medical evaluation by a physician or a health professional (e.g., periodic visits of at least once every year, at least once every six months, at least once every three months, at least once every two months, at least once a month, or at least once a week).
  • Some embodiments of the disclosure further include recording in a subject's medical records that the subject should be administered or prescribed one or more treatment of a pancreatic ⁇ -cell disorder (e.g., any of the exemplary treatments described herein).
  • a subject having an elevated level of soluble MANF as compared to the reference level is selected for pancreatic ⁇ -cell transplantation.
  • the increased risk is relative to a subject that does not have a significant or observable elevation in the level of soluble MANF (e.g., a subject that is not diagnosed as having a pancreatic ⁇ -cell disorder using any of the methods described herein, a healthy subject is not diagnosed as having a disease, or a subject that does not have a symptom of a pancreatic ⁇ -cell disorder or a family history of a pancreatic ⁇ -cell disorder).
  • a subject that does not have a significant or observable elevation in the level of soluble MANF e.g., a subject that is not diagnosed as having a pancreatic ⁇ -cell disorder using any of the methods described herein, a healthy subject is not diagnosed as having a disease, or a subject that does not have a symptom of a pancreatic ⁇ -cell disorder or a family history of a pancreatic ⁇ -cell disorder.
  • the levels of soluble MANF may be determined using standard methods (e.g., any of the antibody-based methods known in the art). The methods can be performed by any health care professional (e.g., a physician, a nurse, a physician's assistant, a laboratory technician, or a nurse's assistant).
  • a health care professional e.g., a physician, a nurse, a physician's assistant, a laboratory technician, or a nurse's assistant.
  • the subjects may present with one or more symptoms of a pancreatic ⁇ -cell disorder (e.g., any of the symptoms of a pancreatic ⁇ -cell disorder described herein).
  • the subject can also be suspected of having a pancreatic ⁇ -cell disorder.
  • the subject can also present with no symptoms or just one symptom of a pancreatic ⁇ -cell disorder.
  • the subject can have a family history of a pancreatic ⁇ -cell disorder (e.g., type 2 diabetes).
  • the methods are performed on a subject that has a detectable or observable pancreatic ⁇ -cell mass and/or has detectable or observable amount pancreatic ⁇ -cell function (e.g., a subject that does not have a complete loss of pancreatic ⁇ -cell mass or pancreatic ⁇ -cell function).
  • Subjects identified as having an increased risk of developing a pancreatic ⁇ -cell disorder may be administered a treatment for a pancreatic ⁇ -cell disorder (e.g., any of the treatments described herein) or may be administered a new or alternative treatment for a pancreatic ⁇ -cell disorder.
  • Subjects identified as having an increased risk of developing a pancreatic ⁇ -cell disorder can also undergo more aggressive therapeutic treatment (e.g., increased periodicity of clinic or hospital visits).
  • Some embodiments of the disclosure further include administering to a subject identified as having an increased risk of developing a pancreatic ⁇ -cell disorder a treatment for a pancreatic ⁇ -cell disorder (e.g., an isolated, purified, or recombinant soluble MANF protein, pioglitazone, GLP-1, or a DPP-4 inhibitor (e.g., sitagliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin, and alogliptin), or any of the compositions described herein, e.g., an therapeutically effective dose of a soluble MANF protein containing a sequence at least 90% identical to SEQ ID NO: 2 and/or apomorphine).
  • a treatment for a pancreatic ⁇ -cell disorder e.g., an isolated, purified, or recombinant soluble MANF protein, pioglitazone, GLP-1,
  • Some embodiments of the disclosure include selecting the subject having an increased risk of developing a pancreatic ⁇ -cell disorder for glucose monitoring (e.g., self-glucose monitoring using a glucometer). Some embodiments of the disclosure further include selecting the subject for periodic medical evaluation by a physician or a health professional (e.g., periodic visits of at least once every year, at least once every six months, at least once every three months, at least once every two months, or at least once a month).
  • a physician or a health professional e.g., periodic visits of at least once every year, at least once every six months, at least once every three months, at least once every two months, or at least once a month.
  • Some embodiments of the disclosure further include recording the results of the test in the subject's medical records, or performing a similar test or any art-known test to determine the risk of developing a pancreatic ⁇ -cell disorder in one or more lineal family members of a subject identified as having an increased risk of developing a pancreatic ⁇ -cell disorder using the methods described herein.
  • the reference level e.g., any of the reference levels of soluble MANF described herein
  • the levels of soluble MANF may be determined using standard molecular biology methods (e.g., any of the antibody-based methods described herein). The methods can be performed by any health care professional (e.g., a physician, a nurse, a physician's assistant, a laboratory technician, or a nurse's assistant).
  • a health care professional e.g., a physician, a nurse, a physician's assistant, a laboratory technician, or a nurse's assistant.
  • the subject may present with one or more symptoms of a pancreatic ⁇ -cell disorder (e.g., any of the symptoms of a pancreatic ⁇ -cell disorder described herein). In some embodiments, the subject can also present with no symptoms or just one symptom of a pancreatic ⁇ -cell disorder. In some embodiments, the subject can have a family history of a pancreatic ⁇ -cell disorder (e.g., type 2 diabetes). In some embodiments, the subject can already be diagnosed as having a pancreatic ⁇ -cell disorder. In some embodiments, the subject is taking a treatment for a pancreatic ⁇ -cell disorder. In some embodiments, the subject is administered a new or alternative treatment for a pancreatic ⁇ -cell disorder during the clinical study.
  • a pancreatic ⁇ -cell disorder e.g., any of the symptoms of a pancreatic ⁇ -cell disorder described herein. In some embodiments, the subject can also present with no symptoms or just one symptom of a pancreatic ⁇ -cell disorder
  • Also provided in the disclosure are methods of detecting endoplasmic reticulum stress in a pancreatic ⁇ -cell that include determining a level of soluble MANF produced by a pancreatic ⁇ -cell, and comparing the level of soluble MANF produced to a reference level (e.g., any of the reference levels of soluble MANF described herein) of soluble MANF.
  • a reference level e.g., any of the reference levels of soluble MANF described herein
  • a decrease or no significant change in the level of soluble MANF produced by the pancreatic ⁇ -cell compared to the reference level of soluble MANF indicates that the pancreatic ⁇ -cell has not experienced a detectable level of endoplasmic reticulum stress.
  • the levels of soluble MANF may be determined using standard molecular biology methods (e.g., any of the antibody-based methods described herein). The methods can be performed by any health care professional (e.g., a physician, a nurse, a physician's assistant, a laboratory technician, or a nurse's assistant) or a scientist.
  • a health care professional e.g., a physician, a nurse, a physician's assistant, a laboratory technician, or a nurse's assistant
  • the pancreatic ⁇ -cell is in a mammal (e.g., a human) and the levels of soluble MANF produced by the pancreatic ⁇ -cell can be determined from a biological sample from the mammal (e.g., a sample containing blood, serum, or plasma).
  • the pancreatic ⁇ -cell can be an endogenous pancreatic ⁇ -cell or a transplanted pancreatic ⁇ -cell (e.g., an autografted, homografted, or xenografted pancreatic ⁇ -cell).
  • the pancreatic ⁇ -cell is an autografted pancreatic ⁇ -cell that has been genetically modified).
  • the pancreatic ⁇ -cells can be present within a pancreas of the mammal or can be present in a tissue other than the pancreas (e.g., the liver). In some embodiments of the disclosure, the pancreatic ⁇ -cells can be present in a device, or a biocompatible material or polymer.
  • the pancreatic ⁇ -cell is present in vitro (e.g., in a tissue culture).
  • a primary pancreatic ⁇ -cell harvested from a mammal can be cultured ex vivo.
  • the cultured pancreatic ⁇ -cell is a primary mammalian (e.g., human, rat, monkey, cow, or pig) pancreatic ⁇ -cell line.
  • the cultured mammalian pancreatic ⁇ -cell can be genetically manipulated (e.g., genetically modified to express one or more proteins or genetically modified to decrease the expression of one or more proteins) or chemically treated (e.g., with one or more growth factors).
  • the pancreatic ⁇ -cell can be cultured in the presence of one or more biocompatible polymers or biosynthetic materials (e.g., polymers or materials that aid in the transplantation of the pancreatic ⁇ -cells into a mammal (e.g., a human)).
  • biocompatible polymers or biosynthetic materials e.g., polymers or materials that aid in the transplantation of the pancreatic ⁇ -cells into a mammal (e.g., a human).
  • biocompatible polymers or biosynthetic materials e.g., polymers or materials that aid in the transplantation of the pancreatic ⁇ -cells into a mammal (e.g., a human).
  • biocompatible polymers or biosynthetic materials e.g., polymers or materials that aid in the transplantation of the pancreatic ⁇ -cells into a mammal (e.g., a human)
  • biocompatible polymers and biosynthetic materials are known in
  • the detection of endoplasmic reticulum stress in pancreatic ⁇ -cells within a subject is followed by one or more of the following: identification of a subject having an increased level of endoplasmic reticulum stress in his or her pancreatic ⁇ -cells, administration of a therapeutic agent (e.g., an agent that will decrease endoplasmic reticulum stress in pancreatic ⁇ -cells, e.g., any of the soluble MANF proteins described herein and/or apomorphine); monitoring of pancreatic ⁇ -cell function (e.g., any of the methods of monitoring pancreatic ⁇ -cell function described herein) in the subject; and increasing the frequency of clinical visits or the level of health monitoring (e.g., increased frequency of blood glucose testing) of the subject.
  • a therapeutic agent e.g., an agent that will decrease endoplasmic reticulum stress in pancreatic ⁇ -cells, e.g., any of the soluble MANF proteins described herein and/or
  • the detection of increased endoplasmic reticulum stress in pancreatic ⁇ -cells in vitro is followed by contacting the pancreatic ⁇ -cell with a therapeutic agent (e.g., an agent that will decrease endoplasmic reticulum stress in pancreatic ⁇ -cells, e.g., any of the soluble MANF proteins described herein or apomorphine) and/or monitoring of pancreatic ⁇ -cell function (e.g., any of the methods of monitoring pancreatic ⁇ -cell function described herein).
  • a therapeutic agent e.g., an agent that will decrease endoplasmic reticulum stress in pancreatic ⁇ -cells, e.g., any of the soluble MANF proteins described herein or apomorphine
  • monitoring of pancreatic ⁇ -cell function e.g., any of the methods of monitoring pancreatic ⁇ -cell function described herein.
  • the pancreatic ⁇ -cell can be cultured in vitro with at least one other cell type or
  • soluble MANF comprising a sequence that is at least 80% identical to SEQ ID NO: 2 and has a biological activity of soluble MANF in methods of treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject that include administering to a subject an effective amount of the soluble MANF (e.g., a purified, isolated, or recombinant soluble MANF protein (e.g., any of the soluble MANF proteins described herein)).
  • soluble MANF e.g., a purified, isolated, or recombinant soluble MANF protein (e.g., any of the soluble MANF proteins described herein)
  • treating can result in a decrease in the number of symptoms of a pancreatic ⁇ -cell disorder (e.g., any of the symptoms described herein) in a subject or a decrease in the severity, intensity, or frequency of one or more symptoms of a pancreatic ⁇ -cell disorder (e.g., any of the symptoms described herein).
  • treating can result in a delay in the onset or one or more symptoms (an increase in the time of actual onset of one or more symptoms in a subject not receiving treatment compared to a subject receiving treatment).
  • treating can result in one or more of the following: a decrease in the blood glucose level(s) in a subject, a decrease in the level of glycated hemoglobin in a subject, a decrease in the rate of loss of the production of insulin in a subject, a decrease in the rate of loss of pancreatic ⁇ -cell function in a subject, and a decrease in the rate of loss of pancreatic ⁇ -cell mass in a subject.
  • the subject does not have erectile dysfunction or Parkinson's disease.
  • soluble MANF administered to a subject contains a sequence that is at least 80% identical (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 2, i.e., to the full length of SEQ ID NO:2, and has a biological activity of a soluble MANF protein (described herein).
  • the soluble MANF administered to a subject contains a sequence that is at least 95% identical (e.g., at least 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 2 (human soluble MANF), i.e., to the full length of SEQ ID NO:2, and has a biological activity of soluble MANF (described herein).
  • the soluble MANF administered to a subject is SEQ ID NO: 2 or an endogenous (wildtype) form of soluble MANF.
  • the soluble MANF can be purified or isolated.
  • the soluble MANF can be a recombinant protein.
  • the comparison of sequences and determination of percent identity between two sequences is accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, J. Mol. Biol., 48:444-453, 1970 ) algorithm, which has been incorporated into the GAP program in the GCG software package (available on the Internet at gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16 and a length weight of 1.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (also available on the Internet at gcg.com), using a NWSgapdna.CMP matrix, a gap weight of 40, and a length weight of 1.
  • percent identity between amino acid sequences referred to herein is determined using the BLAST 2.0 program, which is available to the public at the National Center for Biotechnology Information (NCBI) website. Sequence comparison is performed using an ungapped alignment and using the default parameters (Blossum 62 matrix, gap existence cost of 11, per residue gap cost of 1, and a lambda ratio of 0.85).
  • the mathematical algorithm used in BLAST programs is described in Altschul et al., Nucleic Acids Research 25:3389-3402, 1997 .
  • mammalian forms of soluble MANF have a high degree of sequence identity.
  • biological activity e.g., the ability to treat or delay the onset of a pancreatic ⁇ -cell disorder in a subject, reduce endoplasmic reticulum stress in a pancreatic ⁇ -cell, or reduce or delay endoplasmic reticulum stress-induced apoptotic cell death in population of two or more pancreatic ⁇ -cells
  • residues that are not conserved between various mammalian species of soluble MANF could be altered or removed, while those residues that are highly conserved should not be altered or removed.
  • one skilled in the art can align the various sequences for mammalian soluble MANF proteins provided herein to identify those residues that are highly conserved and those residues that are not conserved.
  • the biological activity of the various forms of soluble MANF can be tested by performing various biological activity assays described herein or those known in the art.
  • the biological activity of a soluble MANF protein can be tested by treating a pancreatic ⁇ -cell cultured in the presence or absence of a soluble MANF (e.g., any of the soluble MANF proteins described herein) and challenging the pancreatic ⁇ -cells with an agent that induces endoplasmic reticulum stress in the pancreatic ⁇ -cells.
  • a soluble MANF having biological activity will reduce the amount of endoplasmic reticulum stress or endoplasmic reticulum stress-induced apoptosis observed in the cells contacted with the soluble MANF and the agent, as compared to the cells not contacted with the soluble MANF and treated with the agent.
  • a soluble MANF having biological activity can reduce one or more markers of endoplasmic reticulum stress in a cell treated with an agent that induces endoplasmic reticulum stress compared to a cell not treated with a soluble MANF and treated with the agent (e.g., reduce the induction of glucose-regulated protein-78 (also known as grp78 or BiP) or bcl-2-associated athanogene-1 (bag-1) expression; reduce activation, Golgi translocation, protease cleavage, or nuclear translocation of activating transcription factor 6 (ATF6); reduce protein kinase RNA-like endoplasmic reticulum kinase (PERK) activation, oligomerization, or autohosphorylation; reduce activation of IRE1; decrease phosphorylation of eIF2I; reduce the intron processing of XBP1 mRNA; reduce activation of a JNK signaling pathway; prevent activation and cleavage of procaspase
  • a soluble MANF administered to the subject can also contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) insertions, additions, deletions, or modifications.
  • a soluble MANF can be covalently attached to a chemical moiety (e.g., a protein (e.g., albumin), a sugar (e.g., N-linked glycans or O-linked glycans, e.g., mannose) (see, e.g., Sola et al., J. Pharm. Sci.
  • the soluble MANF protein used in these methods can also include an HIV tat protein or any other moiety that increases the cellular permeability of the soluble MANF protein.
  • a soluble MANF encoded by a mRNA sequence e.g., an mRNA containing a sequence at least 80% identical (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 3 can be transfected into a bacterial, yeast, or mammalian cell (using a protein expression plasmid or viral vector) that allows for the expression of the soluble MANF by the transfected cell.
  • a mRNA sequence e.g., an mRNA containing a sequence at least 80% identical (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical
  • SEQ ID NO: 3 can be transfected into a bacterial, yeast, or mammalian cell (using a protein expression plasmid or viral vector) that allows for
  • the transfected cells or the culture medium can be collected, and the recombinant soluble MANF protein purified using methods known in the art.
  • a number of additional nucleic acids (mRNA) encoding other mammalian soluble MANF proteins are known in the art.
  • the subject is first identified or selected for treatment using any of the diagnostic methods described herein or any of the methods of predicting a subject at risk of developing a pancreatic ⁇ -cell disorder described herein or known in the art.
  • a subject can be administered at least one (e.g., at least 2, 3, 4, or 5) dose of a soluble MANF (e.g., any of the soluble MANF proteins described herein) and/or apomorphine.
  • a soluble MANF e.g., any of the soluble MANF proteins described herein
  • the soluble MANF and/or apomorphine can be administered to the subject at least once a day (e.g., twice a day, three times a day, and four times a day), at least once a week (e.g., twice a week, three times a week, four times a week), and/or at least once a month.
  • a subject can be treated (e.g., periodically administered a soluble MANF) for a prolonged period of time (e.g., at least one month, two months, six months, one year, two years, three years, four years, or five years).
  • a prolonged period of time e.g., at least one month, two months, six months, one year, two years, three years, four years, or five years.
  • the dosage of the soluble MANF and/or apomorphine to be administered to the subject can be determined by a physician by consideration of a number of physiological factors including, but not limited to, the sex of the subject, the weight of the subject, the age of the subject, and the presence of other medical conditions.
  • the soluble MANF and/or apomorphine can be administered to the subject orally, intravenously, intraarterially, subcutaneously, intramuscularly, intracranially, or via injection into the cerebrospinal fluid.
  • the agent may be formulated as a solid (e.g., for oral administration) or a physiologically acceptable liquid carrier (e.g., saline) (e.g., for intravenous, intraarterial, subcutaneous, intramuscular, or cerebrospinal administration).
  • the subject is further administered at least one (e.g., two, three, four, or five) other treatment of a pancreatic ⁇ -cell disorder (e.g., any of the treatments for a pancreatic ⁇ -cell disorder described herein, e.g., any of the insulins described herein).
  • the soluble MANF and/or apomorphine is formulated together with at least one (e.g., two, three, or four) other treatment of a pancreatic ⁇ -cell disorder (e.g., formulated in a physiologically acceptable buffer or medium for systemic administration) (e.g., any of the pharmaceutical compositions described herein).
  • pancreatic ⁇ -cell methods of reducing endoplasmic reticulum stress in a pancreatic ⁇ -cell. These methods include contacting the pancreatic ⁇ -cell with an effective amount of one or more (e.g., two or three) of soluble MANF (e.g., any of the soluble MANF proteins described herein, e.g., a recombinant, purified, or isolated soluble MANF protein containing a sequence at least 80% identical to SEQ ID NO: 2), apomorphine, and pioglitazone.
  • the pancreatic ⁇ -cell is in vitro (tissue culture).
  • the pancreatic ⁇ -cell can be in a subject.
  • the pancreatic ⁇ -cell used in these experiments can be any pancreatic ⁇ -cell described herein.
  • the pancreatic ⁇ -cell can be contacted with one or more (e.g., two or three) of a soluble MANF, apomorphine, and pioglitazone for an extended period of time (e.g., at least 15 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 8 hours, 10 hours, 12 hours, or 24 hours).
  • the pancreatic ⁇ -cell is contacted with several doses of one or more (e.g., two or three) of a soluble MANF, apomorphine, and pioglitazone (e.g., at least two doses, three doses, four doses, or five doses), e.g., at regular, timed intervals (e.g., approximately once a day, once a week, or once a month).
  • a soluble MANF e.g., a soluble MANF, apomorphine, and pioglitazone
  • timed intervals e.g., approximately once a day, once a week, or once a month.
  • a reduction in endoplasmic reticulum stress can be determined using any methods known in the art for detecting endoplasmic reticulum stress in a cell (e.g., detecting or assessing any of the markers of endoplasmic reticulum stress described herein). For example, a reduction in endoplasmic reticulum stress can be detected by a reduction in one or more markers of endoplasmic reticulum stress in a cell.
  • a reduction in endoplasmic reticulum stress can be observed by one or more of the following events: a reduction in the induction of grp78 (BiP) or bag-1 expression; a reduction in the activation, Golgi translocation, protease cleavage, or nuclear translocation of ATF6; a reduction in PERK activation, oligomerization, or autohosphorylation; a reduction in the activation of IRE1; a reduction in phosphorylation of eIF2I; a reduction in the intron processing of XBP1 mRNA; a reduction in the activation of a JNK signaling pathway; a reduction in the activation and cleavage of procaspase 4; and a reduction in the shift in the redox environment of the endoplasmic reticulum induced by exposure to a ER-stress inducing agent (e.g., as compared to a control pancreatic ⁇ -cell exposed to a ER-
  • a reduction in the shift in the redox environment of the endoplasmic reticulum can be measured using redox-sensitive dyes or proteins, e.g., the reporter protein described in the Examples.
  • the reduction in endoplasmic reticulum stress in a pancreatic ⁇ -cell can be compared to the amount of endoplasmic reticulum stress observed or detected in a pancreatic ⁇ -cell not contacted with one or more of soluble MANF, apomorphine, and pioglitazone, respectively (e.g., in vitro or in a subject).
  • the reduction in endoplasmic reticulum stress in a pancreatic ⁇ -cell is relative to a control pancreatic ⁇ -cell that is not contacted with a soluble MANF protein, apomorphine, or pioglitazone, but is contacted with an agent that induces endoplasmic reticulum stress (e.g., thapsigargin).
  • an agent that induces endoplasmic reticulum stress e.g., thapsigargin
  • a health care professional e.g., any health care professional described herein
  • a scientist e.g., any scientist described herein
  • Pancreatic ⁇ -cells having endoplasmic reticulum stress can activate apoptotic pathways within the cell.
  • methods of reducing or delaying endoplasmic reticulum stress-induced apoptosis in a population of two or more pancreatic ⁇ -cells that include contacting the population of pancreatic ⁇ -cells with an effective amount of one or more (e.g., two or three) of a soluble MANF (e.g., any of the soluble MANF proteins described herein), apomorphine, and pioglitazone.
  • a soluble MANF e.g., any of the soluble MANF proteins described herein
  • the pancreatic ⁇ -cell is in vitro (tissue culture).
  • the pancreatic ⁇ -cell can be in a subject (e.g., in an engrafted biocompatible material or polymer).
  • the pancreatic ⁇ -cells used in these methods can be any pancreatic ⁇ -cells described herein.
  • the pancreatic ⁇ -cell can be contacted with one or more (e.g., two or three) of a soluble MANF, apomorphine, and pioglitazone for an extended period of time (e.g., at least 15 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 8 hours, 10 hours, 12 hours, or 24 hours).
  • the pancreatic ⁇ -cell is contacted with several doses of one or more (e.g., two or three) of a soluble MANF, pioglitazone, and apomorphine (e.g., at least two doses, three doses, four doses, or five doses), e.g., at regular timed intervals.
  • a soluble MANF e.g., a soluble MANF, pioglitazone, and apomorphine
  • apomorphine e.g., at least two doses, three doses, four doses, or five doses
  • the onset and timing of apoptosis in a pancreatic ⁇ -cell population can be determined using any of the methods described herein or those known in the art.
  • Contacting a pancreatic ⁇ -cell population with one or more (e.g., two or three) of a soluble MANF, apomorphine, and pioglitazone can mediate a decrease in the percentage of pancreatic ⁇ -cells within the population that undergo apoptosis (e.g., endoplasmic reticulum stress-induced apoptosis) or delay the onset of apoptosis within the population of pancreatic ⁇ -cells.
  • apoptosis e.g., endoplasmic reticulum stress-induced apoptosis
  • the decrease or delay in endoplasmic reticulum stress-induced apoptosis in cells treated with one or more (e.g., two or three) of a soluble MANF, apomorphine, and pioglitazone can be compared to a population of pancreatic ⁇ -cells that are not treated with one or more (e.g., two or three) of a soluble MANF, apomorphine, and pioglitazone.
  • the decrease or delay in endoplasmic reticulum stress-induced apoptosis in cells treated with one or more (e.g., two or three) of a soluble MANF, apomorphine, and pioglitazone can be compared to a control population of pancreatic ⁇ -cells not treated with one or more (e.g., two or three) of a soluble MANF, apomorphine, and pioglitazone, but contacted with an agent that induces endoplasmic reticulum stress (e.g., thapsigargin).
  • an agent that induces endoplasmic reticulum stress e.g., thapsigargin
  • Methods for detecting apoptotic cell death include, without limitation, the cleavage of cellular caspases (e.g., procaspase-3 and procaspase-4), Hoescht and 7-amino-actinomycin uptake, TdT-mediated dUTP nick end labeling assay, and annexin membrane staining.
  • cellular caspases e.g., procaspase-3 and procaspase-4
  • Hoescht and 7-amino-actinomycin uptake e.g., procaspase-3 and procaspase-4
  • Hoescht and 7-amino-actinomycin uptake e.g., TdT-mediated dUTP nick end labeling assay
  • annexin membrane staining e.g., annexin membrane staining.
  • compositions that contain at least soluble MANF comprising a sequence that is at least 80% identical to SEQ ID NO: 2 and has a biological activity of soluble MANF (e.g., any of the soluble MANF proteins described herein, e.g., a purified, isolated, or recombinant soluble MANF);and apomorphine, and/or at least one other treatment for a pancreatic ⁇ -cell disorder (e.g., one or more of any of the treatments of a pancreatic ⁇ -cell disorder described herein, e.g., pioglitazone, TUDCA, and any of the insulins described herein).
  • soluble MANF e.g., any of the soluble MANF proteins described herein, e.g., a purified, isolated, or recombinant soluble MANF
  • apomorphine e.g., one or more of any of the treatments of a pancreatic ⁇ -cell disorder described herein, e
  • the compositions are formulated with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions and formulations can be administered parenterally, orally or by local administration, such as by aerosol or transdermally.
  • the pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005 .
  • compositions provided herein may be formulated for administration, in any convenient way for use in human or veterinary medicine.
  • Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the compositions.
  • Formulations of the compositions of the invention include those suitable for intradermal, inhalation, oral/ nasal, topical, and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient e.g., soluble MANF and/or apomorphine, and one or more additional therapeutic agents of a pancreatic ⁇ -cell disorder
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect (e.g., one or more of any of the therapeutic effects described herein).
  • compositions of this invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals.
  • Such drugs can contain sweetening agents, flavoring agents, coloring agents, and preserving agents.
  • a formulation can be admixed with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture.
  • Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc., and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
  • Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
  • Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen.
  • Disintegrating or solubilizing agents may be added, such as the crosslinked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Push-fit capsules can contain active agents mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • a filler or binders such as lactose or starches
  • lubricants such as talc or magnesium stearate
  • stabilizers optionally, stabilizers.
  • the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • Aqueous suspensions can contain an active agent (e.g., a soluble MANF and/or apomorphine, and one or more additional treatments of a pancreatic ⁇ -disorder) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections.
  • an active agent e.g., a soluble MANF and/or apomorphine, and one or more additional treatments of a pancreatic ⁇ -disorder
  • Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, and dispersing or wetting agents such as a naturally-occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long-chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene
  • the aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, or saccharin.
  • preservatives such as ethyl or n-propyl p-hydroxybenzoate
  • coloring agents such as a coloring agent
  • flavoring agents such as aqueous suspension
  • sweetening agents such as sucrose, aspartame, or saccharin.
  • Formulations can be adjusted for osmolality.
  • oil-based pharmaceuticals are used for administration.
  • Oil-based suspensions can be formulated by suspending active agents in a vegetable oil, such as arachis oil, olive oil, sesame oil, or coconut oil, or in a mineral oil, such as liquid paraffin; or a mixture of these. See, e.g., U.S. Patent No. 5,716,928 , describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also, U.S. Patent No. 5,858,401 ).
  • the oil suspensions can contain a thickening agent, such as beeswax, hard paraffin, or cetyl alcohol.
  • Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol, or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid.
  • an injectable oil vehicle see Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997 .
  • compositions can also be in the form of oil-in-water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these.
  • Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally-occurring phosphatides, such as soybean lecithin, esters, or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate.
  • the emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs.
  • Such formulations can also contain a demulcent, a preservative, or a coloring agent.
  • these injectable oil-in-water emulsions of the invention comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate, and/or an ethoxylated sorbitan trioleate.
  • the pharmaceutical compounds can also be administered by in intranasal or intraocular routes including insufflation, powders, and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi, J. Clin. Pharmacol. 35:1187-1193, 1995 ; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111, 1995 ).
  • the pharmaceutical compounds can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • the pharmaceutical compounds can also be delivered as microspheres for slow release in the body.
  • microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995 ; as biodegradable and injectable gel formulations, see, e.g., Gao, Pharm. Res. 12:857-863, 1995 ; or, as microspheres for oral administration, see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997 .
  • the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV), intramuscular, intraperitoneal, or subcutaneous administration, or administration into a body cavity, a lumen of an organ, or into the cerebrospinal fluid of a subject.
  • IV intravenous
  • These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier.
  • Acceptable vehicles and solvents that can be employed are water and Ringer's solution, or an isotonic sodium chloride.
  • sterile fixed oils can be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can likewise be used in the preparation of injectables.
  • formulations may be sterilized by conventional, well known sterilization techniques.
  • the formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.
  • concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs.
  • the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
  • This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3-butanediol.
  • the administration can be by bolus or continuous (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).
  • the pharmaceutical compounds and formulations can be lyophilized.
  • Stable lyophilized formulations comprising a soluble MANF and/or apomorphine, and one or more additional treatments of a pancreatic ⁇ -cell disorder can be made by lyophilizing a solution comprising a soluble MANF and/or apomorphine, and the one or more additional treatments and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose, or mixtures thereof.
  • a bulking agent e.g., mannitol, trehalose, raffinose, and sucrose, or mixtures thereof.
  • a process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5, but less than 6.5. See, e.g., US2004/0028670 .
  • compositions and formulations can be delivered by the use of liposomes.
  • liposomes particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400 and 6,007,839 ; Al-Muhammed, J. Microencapsul. 13:293-306, 1996 ; Chonn, Currr. Opin. Biotechnol. 6:698-708, 1995 ; and Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989 .
  • compositions of the invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a subject who is at risk of or has a disorder described herein, in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its complications; this can be called a therapeutically effective amount.
  • pharmaceutical compositions of the invention are administered in an amount sufficient to reduce the number of symptoms or reduce the severity, duration, or frequency of one or more symptoms of a pancreatic ⁇ -cell disorder in a subject.
  • the amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose.
  • the dosage schedule and amounts effective for this use i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
  • the dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones, J. Steroid Biochem. Mol. Biol. 58:611-617, 1996 ; Groning, Pharmazie 51:337-341, 1996 ; Fotherby, Contraception 54:59-69, 1996 ; Johnson, J. Pharm. Sci. 84:1144-1146, 1995 ; Rohatagi, Pharmazie 50:610-613, 1995 ; Brophy, Eur. J. Clin. Pharmacol.
  • formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, and the like.
  • the formulations should provide a sufficient quantity of the active agents to effectively treat, prevent or ameliorate conditions, diseases, or symptoms.
  • pharmaceutical formulations for oral administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day.
  • Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ.
  • Substantially higher dosages can be used in topical or oral administration or administering by powders, spray, or inhalation.
  • Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21st ed., 2005 .
  • kits that contain at least one antibody or antigen-binding antibody fragment (e.g., Fab, F(ab') 2 , Fab', scFv, di-scFv, or sdAb) that specifically binds to a soluble MANF protein (e.g., any of the soluble MANF proteins described herein) and at least one (e.g., two, three, or four) antibody or antigen-binding antibody fragment that specifically binds to one other marker of a pancreatic ⁇ -cell disorder (e.g., insulin, C-protein, and IAPP).
  • a pancreatic ⁇ -cell disorder e.g., insulin, C-protein, and IAPP
  • the antibodies or antigen-binding antibody fragments included in the kits are localized on a substrate (e.g., an enzyme-linked immunosorbent assay).
  • the kits can further include an isolated, purified, or recombinant soluble MANF protein (e.g., any of the soluble MANF proteins described herein).
  • one or more of the antibodies or antigen-binding antibody fragments is/are labeled (e.g., a radioisotope, a fluorophore, or a binding protein (e.g., avidin)).
  • kits can be useful for, e.g., for diagnosing a pancreatic ⁇ -cell disorder, identifying a subject at risk of developing a pancreatic ⁇ -cell in a subject, or Monitoring pancreatic ⁇ -cell function or pancreatic ⁇ -cell mass in a subject.
  • the kits can further contain instructions for performing any of the methods described herein.
  • reporter proteins that contain a binding protein (BiP) signal sequence (e.g., a mouse or a human BiP signal sequence), a redox-sensitive fluorescent protein (e.g., a redox-sensitive green fluorescent protein and a redox-sensitive yellow fluorescent protein), and the amino acid sequence KDEL (Lys-Asp-Glu-Leu).
  • BiP binding protein
  • a redox-sensitive fluorescent protein e.g., a redox-sensitive green fluorescent protein and a redox-sensitive yellow fluorescent protein
  • the amino acid sequence KDEL Lithys-Asp-Glu-Leu
  • the BiP signal sequence is at the N-terminus
  • the redox-sensitive fluorescent protein is C-terminal to the BiP signal sequence
  • the amino acid sequence KDEL is C-terminal to the redox-sensitive fluorescent protein.
  • there are 1 to 100 amino acids e.g., 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, and 1 to 10 amino acids
  • the reporter protein e.g., BiP signal sequence, the redox-sensitive fluorescent protein, and the KDEL sequence.
  • the amino acid sequence of a redox-sensitive green fluorescent protein is listed below.
  • the reporter proteins described herein can contain a sequence at least 80% identical (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to SEQ ID NO: 10 (as long as the resulting protein maintains its redox-sensitive fluorescence properties).
  • mutations introduced into a redox-sensitive green fluorescent protein or a redox-sensitive yellow fluorescent protein should not include mutations in the cysteines.
  • the redox-sensitive fluorescent protein is a redox-sensitive yellow fluorescent protein (e.g., rxYFP; described in Ostergaard et al., EMBO J. 20:5853-5862, 2001 ).
  • the reporter proteins used in the methods described herein can contain any mammalian BiP signal sequence (e.g., a human or mouse BiP signal sequence).
  • the reporter protein and nucleic acids encoding these reporter proteins provides a means for sensitive (e.g., significantly improved) detection of subtle fluctuations in the redox environment within an intact pancreatic ⁇ -cell.
  • the reporter protein contains a sequence that is at least 80% identical (e.g., at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to a reporter protein containing the sequence of SEQ ID NO: 14 (shown below).
  • the reporter protein is encoded by a nucleic acid containing a sequence that is at least 80% identical (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 15.
  • mutations in the reporter protein of SEQ ID NO: 14 should not include mutations in the cysteines.
  • SEQ ID NO: 14 can be tested using any of the methods described herein to determine whether the mutants maintain their redox-dependent fluorescence properties.
  • the specific redox-dependent fluorescence properties of a protein containing a sequence at least 80% identical to SEQ ID NO: 14 (MEROS-GFP) are described in detail in the Examples.
  • These methods include providing a pancreatic ⁇ -cell, contacting the pancreatic ⁇ -cell with a candidate compound, and determining the level of soluble MANF produced by the pancreatic ⁇ -cell in the presence of the candidate compound, and comparing the level of soluble MANF produced by the pancreatic ⁇ -cell to a reference level of soluble MANF.
  • an elevated level of soluble MANF produced by the pancreatic ⁇ -cell compared to the reference level indicates that the test compound may be useful for one or more of the following: treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject, decreasing endoplasmic reticulum stress in a pancreatic ⁇ -cell, and reducing or delaying endoplasmic reticulum stress-induced apoptotic cell death in pancreatic ⁇ -cells.
  • the pancreatic ⁇ -cell(s) used in these methods can be any of the pancreatic ⁇ -cells described herein (e.g., a pancreatic ⁇ -cell line (e.g., any of the pancreatic ⁇ -cell lines described herein) or primary pancreatic ⁇ -cells).
  • the levels of soluble MANF may be determined using standard molecular biology methods (e.g., any of the antibody-based methods described herein). The methods can be performed by any health care professional (e.g., a physician, a nurse, a physician's assistant, a laboratory technician, or a nurse's assistant) or a scientist.
  • a health care professional e.g., a physician, a nurse, a physician's assistant, a laboratory technician, or a nurse's assistant
  • the reference level is a level of soluble MANF produced by a pancreatic ⁇ -cell in the absence of the candidate compound. In some embodiments, the reference level is a level of soluble MANF present in a subject that does not have a pancreatic ⁇ -cell disorder, does not have a symptom of a pancreatic ⁇ -cell disorder, or a family history of a pancreatic ⁇ -cell disorder. In some embodiments of the disclosure, the reference level is a level of soluble MANF produced in a primary pancreatic ⁇ -cell from a mammal or a mammalian pancreatic ⁇ -cell line. In some embodiments, the reference level is a threshold level of soluble MANF.
  • a mammalian cell e.g., a mammalian pancreatic ⁇ -cell or pancreatic ⁇ -cell line
  • a reporter protein containing from a BiP signal sequence, a redox-sensitive fluorescent protein, and the amino acid sequence KDEL e.g., a mammalian pancreatic ⁇ -cell or pancreatic ⁇ -cell line
  • contacting the cell with a test compound determining the amount of oxidized reporter protein present in the cell
  • comparing the amount of oxidized reporter protein present in the cell compared to a reference level where an elevated level of oxidized reporter protein in the cell compared to the reference level indicates that the candidate compound may be useful for treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject, decreasing endoplasmic reticulum stress in a pancreatic ⁇ -cell, and/or reducing or delaying endoplasmic reticulum stress-induced apoptotic cell death in pancreatic ⁇ -cells.
  • the reference level is the amount of the oxidized reporter protein present in a mammalian cell in the absence of the candidate agent.
  • the cell is contacted with both the candidate agent and an agent that induces ER stress and the reference level is a level of oxidized reporter protein present in a cell treated with the agent that induces ER stress alone.
  • agents that induce ER stress are described herein. Additional examples of agents that induce ER stress are known in the art.
  • the reference level is a threshold level of oxidized reporter protein.
  • the cells used can be human, mouse, rat, pig, monkey, or bovine cells.
  • the cells can be any pancreatic ⁇ -cell line described herein or known in the art.
  • the reporter protein is SEQ ID NO: 14, or a protein containing a sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 14.
  • the methods include providing a mammalian cell (e.g., a mammalian pancreatic ⁇ -cell) expressing a reporter protein containing from a BiP signal sequence, a redox-sensitive fluorescent protein, and the amino acid sequence KDEL; contacting the cell with a test compound; determining the amount of reduced reporter protein present in the cell; and comparing the amount of reduced reporter protein present in the cell compared to a reference level; where an increased level of reduced reporter protein in the cell compared to the reference level indicates that the candidate compound may be useful for treating or delaying the onset of a pancreatic ⁇ -cell disorder in a subject, decreasing endoplasmic reticulum stress in a pancreatic ⁇ -cell, and/or reducing or delaying endoplasmic reticulum stress-induced apoptotic cell death in pancreatic ⁇ -cells.
  • a mammalian cell e.g., a mammalian pancreatic ⁇ -cell
  • the reference level is the amount of the reduced reporter protein present in a mammalian cell in the absence of the candidate agent.
  • the cell is contacted with both the candidate agent and an agent that induces ER stress and the reference level is a level of reduced reporter protein present in a cell treated with the agent that induces ER stress alone.
  • agents that induce ER stress are described herein. Additional examples of agents that induce ER stress are known in the art.
  • the reference level is a threshold level of reduced reporter protein.
  • the cells used can be human, mouse, rat, pig, monkey, or bovine cells.
  • the cells can be any pancreatic ⁇ -cell line described herein or known in the art.
  • the reporter protein is SEQ ID NO: 14, or a protein containing a sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 14.
  • the determining is performed by detecting one or more fluorescence properties of the reporter protein in the cell (e.g., detecting spectral features that are unique for the reduced or oxidized form of the reporter protein).
  • the level of the reduced or oxidized form of the reporter protein is determined using a fluorescent plate reader or using fluorescence-assisted cell sorting (FACS).
  • pancreatic ⁇ -cells e.g., INS-1 832/13
  • the reporter protein can be plated into 6-well plates, contacted with a candidate compound, and then harvested by trypsinization. After washing with phosphate buffered saline, the cells can be suspended in a suitable medium (e.g., 11 mM glucose-Hanks buffered salt solution), and FACS performed with LSRII (BD) to determine the levels of the reduced or oxidized form of the reporter protein present in the cell.
  • a suitable medium e.g., 11 mM glucose-Hanks buffered salt solution
  • a fluorescent plate reader can be used to determine the level of the reduced or oxidized form of the reporter protein present in the cell.
  • a pancreatic ⁇ -cell line e.g., INS-1 832/13 cells
  • the levels of the reduced or oxidized form of the reporter protein can be detected using the fluorescent plate reader.
  • the substraction of background signal should be performed prior to determining the level of the reduced or oxidized form of the reporter protein.
  • the reporter protein contains SEQ ID NO: 14 or a protein containing a sequence that is at least 80% identical to SEQ ID NO: 14.
  • the reduced form of the reporter protein has an excitation wavelength of 473 nm and an emission wavelength of 510 nm
  • the oxidized form of the reporter protein has an excitation wavelength of 394 nm and an emission wavelength of 510 nm.
  • a ratio of the level of the reduced form of the reporter protein to the level of the oxidized form of the reporter protein in the cell can be determined.
  • the calculated ratio can be compared to a reference ratio.
  • the reference ratio can be the ratio from a cell that is not treated with a candidate agent.
  • the reference ratio can also be a threshold ratio.
  • the cell is contacted with a candidate agent and an agent that induces ER stress, the ratio of the level of the reduced form of the reporter protein to the level of the oxidized form of the reporter protein in the cell is determined, and the ratio in the cell is compared to a reference ratio from a cell treated with the agent that induces ER stress alone.
  • a candidate agent that decreases the ratio in the cell as compared to the reference ratio is identified as a candidate agent for treating a pancreatic ⁇ -cell disorder in a subject.
  • Some embodiments of the above methods further include testing the candidate compound in an animal model of a pancreatic ⁇ -cell disorder (e.g., determining whether administration of the candidate compound will treat (e.g., reduce the severity, frequency, or duration) one or more symptoms of a pancreatic ⁇ -cell disorder in an animal model or delay the onset of one or more symptoms of a pancreatic ⁇ -cell disorder in an animal model).
  • Non-limiting animal models of type 2 diabetes include Zucker fatty rats (ZFR), ob/ob (obese) mice, cp (corpulent) rats, Zucker diabetic fatty (ZDF) rats, sand rats ( Psammomys obesars ), obsess rhesus monkeys, KK mice, and KK-A y mice (described in Srinivasan et al., Indian J. Med. Res. 125:451-472, 2007 ).
  • Non-limiting animal models of type 1 diabetes include non-obese diabetic (NOD) mice and bio breeding (BB) rats (described in Rees et al., Diabetic Med. 22:359-370, 2005 ).
  • NOD non-obese diabetic
  • BB bio breeding
  • Some embodiments of the above methods further include testing whether the candidate compound will decrease or delay endoplasmic reticulum stress-induced apoptotic cell death in a population of pancreatic ⁇ -cells (e.g., a reduction or delay in endoplasmic reticulum stress-induced apoptotic cell death in a population of pancreatic ⁇ -cells treated with the candidate agent and an agent that induces endoplasmic reticulum stress compared to a population of pancreatic ⁇ -cells treated with the agent that induces endoplasmic reticulum stress in the absence of the candidate agent).
  • endoplasmic reticulum stress-induced apoptotic cell death in a population of pancreatic ⁇ -cells e.g., a reduction or delay in endoplasmic reticulum stress-induced apoptotic cell death in a population of pancreatic ⁇ -cells treated with the candidate agent and an agent that induces endoplasmic reticulum stress compared to a population of
  • Methods for detecting apoptotic cell death include, without limitation, the cleavage of cellular caspases (e.g., procaspase-3 and procaspase-4), Hoescht and 7-amino-actinomycin uptake, TdT-mediated dUTP nick end labeling assay, and annexin membrane staining.
  • cellular caspases e.g., procaspase-3 and procaspase-4
  • Hoescht and 7-amino-actinomycin uptake e.g., procaspase-3 and procaspase-4
  • Hoescht and 7-amino-actinomycin uptake e.g., TdT-mediated dUTP nick end labeling assay
  • annexin membrane staining e.g., annexin membrane staining.
  • Some embodiments of the above methods further include testing whether the candidate compound prevents or delays the induction of other markers of endoplasmic reticulum stress in a pancreatic ⁇ -cell (e.g., whether the candidate compound reduces the induction of gip78 (BiP) or bag-1 expression; reduces activation, Golgi translocation, protease cleavage, or nuclear translocation of ATF6; reduces PERK activation, oligomerization, or autohosphorylation; reduce activation of IRE1; decreases phosphorylation of eIF2I; reduces the intron processing of XBP1 mRNA; reduces activation of a JNK signaling pathway; prevents activation and cleavage of procaspase 4; and/or prevents or decrease the shift in the endoplasmic reticulum redox environment (e.g., measured using any of the reporter proteins described herein)).
  • the candidate compound reduces the induction of gip78 (BiP) or
  • Expression levels of BiP and Bag-1 can be determined using quantitative real-time PCR with sets of primers that are designed to hybridize to portions of BiP or Bag-1. Expression levels or the processing, activation, phosphorylation, or cellular localization of BiP, Bag-1, PERK, IRE1, eIF2I, XBP1, and ATF6 can also be determined using antibodies that specifically bind to one BiP, Bag-1, PERK, IRE1, eIF2I, XBP1, or ATF6 using methods known in the art.
  • the prevention or delay in one or more markers of endoplasmic reticulum stress is cells treated with the candidate agent and an agent that induces endoplasmic reticulum stress is compared to pancreatic ⁇ -cell(s) treated with the agent that induces endoplasmic reticulum stress in the absence of the candidate agent.
  • a candidate compound can be a protein, a peptide, a nucleic acid (e.g., RNA or DNA), an inorganic compound, a lipid, an oligosaccharide, or any combination thereof.
  • Libraries of candidate compounds that can be used in the above methods are commercially available.
  • the candidate compounds to be screened can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds ( Lam, Anticancer Drug Des., 12:145, 1997 ).
  • Libraries of compounds may be presented in solution (e.g., Houghten, Bio/Techniques, 13:412-421, 1992 ), or on beads ( Lam, Nature, 354:82-84, 1991 ), chips ( Fodor, Nature 364:555-556, 1993 ), bacteria ( U.S. Pat. No. 5,223,409 ), spores ( U.S. Pat. Nos. 5,571,698 ; 5,403,484 ; and 5,223,409 ), plasmids ( Cull et al., Proc. Natl. Acad. Sci.
  • INS-1 832/13 and MIN6 rodent pancreatic ⁇ -cell lines
  • INS-1 832/13 cells were cultured in RPMI-1640 containing 10% fetal bovine serum, penicillin, streptomycin, sodium pyruvate, and 0.1% ⁇ -mercaptoethanol.
  • Primary islets were obtained from Prodo, and plated into 6-well plates precoated with laminin V produced by 804G cells, and cultured in CMRL medium supplemented with fetal bovine serum, non-essential amino acids, sodium pyruvate, and antibiotics.
  • Soluble MANF protein was detected in the medium of a cultured rat pancreatic ⁇ -cell line (INS-1 832/13) following treatment with thapsigargin (50 nM) ( Figure 1 ). Elevated levels of MANF mRNA were detected in the same cell line following treatment with either 5 ⁇ M tunicamycin or 20 nM thapsigargin for 24 hours ( Figure 2 ).
  • Example 2 System for monitoring redox states in the ER
  • MEROS-GFP displayed distinct excitation spectra in the fully oxidized and reduced species in NSC34 cells, with maxima at 394 nm and 473 nm ( Figure 10A ).
  • NSC 34 cells were cultured in DMEM containing 10% fetal bovine serum, penicillin, and streptomycin.
  • the ratio between fluorescence from excitation 473 nm versus 394 nm normalized to wild-type untreated cells is called the MEROS-GFP ratio.
  • the MEROS-GFP ratio was determined using a fluorescent plate reader. In these experiments, the INS-1 832/13 cells were plated onto a 96-well plate at 50,000 cells/well, the cells treated with H 2 O 2 or DTT at various concentrations for 30 minutes, and the fluorescence at excitation wavelength 473 nm and emission wavelength 510 nm (for reduced MEROS-GFP) or at excitation wavelength 394 nm and emission wavelength 510 nm (for oxidized MEROS-GFP) was measured. The MEROS-GFP ratio was determined after substraction of background signal.
  • INS-1 832/13 cells left untreated or treated with 2 mM DTT were lysed with 1x SDS-PAGE sample buffer containing 25 mM AMS with or without 2- ⁇ -mercaptoethanol, boiled at 95 °C for 10 minutes, electrophoresed using SDS-PAGE, and immunoblotted using an anti-GFP antibody.
  • SDS-PAGE sample buffer containing 25 mM AMS with or without 2- ⁇ -mercaptoethanol boiled at 95 °C for 10 minutes
  • electrophoresed using SDS-PAGE immunoblotted using an anti-GFP antibody.
  • non-reducing SDS-PAGE of lysates from DTT-treated cells showed only one slower migrating form of MEROS-GFP, indicating that the DTT treatment fully reduced MEROS-GFP in vivo ( Figure 13 ).
  • MEROS-GFP ratio was also monitored in DTT-treated cells at different time points.
  • Figure 14 shows that the ER could be reduced within a few minutes of treatment of DTT, and return to an oxidized environment within a minute of DTT washout.
  • the median MEROS-GFP ratio was also increased by both experimental and physiological inducers of ER stress, including tunicamycin, thapsigargin, brefeldin A, MG 132 ( Figures 19 and 20 ), chronic high glucose ( Figure 21 ), serum depletion, glucose deprivation ( Figure 22 ), palmitate, human islet amyloid polypeptide (hIAPP), and inflammatory cytokines ( Figures 23 and 24 ).
  • This BiP-mCherry reporter plasmid was transfected into INS-1 832/13 cells expressing MEROS-GFP, and FACS analysis was used to monitor the MEROS-GFP ratio and the activation levels of the UPR via BiP-mCherry in the same cells ( Figure 31 ). The activation levels of Bit-cherry and the MEROS-GFP ratio were monitored following the induction of ER stress.
  • INS-1 832/13 cells expressing MEROS-GFP were plated onto 6-well plates, treated with each compound for indicated times, and then harvested by trypsinization. After washing with phosphate buffered saline, cells were resuspended in the 11 mM glucose-Hanks buffered salt solution. Flow cytometry analyses were performed with LSRII (BD).
  • Example 5 A small molecule screen for compounds that shift ER from a reducing to an oxidizing environment
  • Pioglitazone was shown to shift the ER to an oxidizing environment in pancreatic ⁇ -cells treated with thapsigargin ( Figure 38 , right panel) and protect these cells from cell death ( Figure 38 , left panel).
  • Another small molecule, TUDCA has been used for treatment of gallstones and biliary cirrhosis and shown to mitigate ER stress in mouse models of diabetes. TUDCA was shown to also shift the ER towards an oxidizing environment ( Figure 38 , right panel) and to protect cells from death under ER stress conditions ( Figure 38 , left panel).
  • a second screen was performed using 0.4 mM palmitate in combination with 20 mM glucose to induce ER stress. Between both screens, 9 common positive compounds were identified, of which 5 were eliminated due to autofluorescence. To further eliminate false positives, INS-1 832/13 cells stably-expressing MEROS-GFP were pretreated with the remaining 4 common compounds, challenged with 0.5 mM palmitate for 24 hours, and the MEROS-GFP ratio measured using FACS. This additional step removed two compounds as false positives. The remaining two compounds were the clinically used agents apomorphine and griseofulvin. Although positively identified in the screening assays, griseofulvin had strong toxic effects on INS-1 832/13 cells.
  • Example 6 Apomorphine shifts the ER toward an oxidizing environment and confers protection against ER stress
  • INS-1 832/13 cells expressing MEROS-GFP were treated with apomorphine for 24 hours, and then challenged with palmitate for 24 hours.
  • the data show that apomorphine treatment decreased the population of cells that had reduced ER ( Figure 40 ).
  • Cell viability and mitochondrial membrane potential in INS-1 832/13 cells treated with palmitate was measured using propidium iodide (PI) and MitoProbe (Invitrogen) staining, respectively.
  • PI propidium iodide
  • MitoProbe Invitrogen
  • INS-1 832/13 cells from ER stress-mediated cell death induced by a strong ER stress inducer, thapsigargin ( Figure 43 ).
  • thapsigargin a strong ER stress inducer
  • Example 7 Small molecules shifting the ER towards an oxidizing environment can alleviate the pathology of cellular models of ER stress
  • Example 8 Soluble MANF protects pancreatic ⁇ -cells from ER stress and ER stress-induced apoptotic cell death
  • the cells were then analyzed using FACS analysis using an excitation spectrum between 460-495 nm and an emission spectrum of between 520-570 nm (FITC-A optical filter) which allows for the specific detection of fluorescent emission from reduced EroGFP in the transfected cells.
  • the data show that treatment with soluble MANF results in a decrease in the number of cells containing a detectable level of reduced MEROS-GFP ( Figure 45 ; lower right panel vs. lower left panel). Consistent with the data above, these data show that treatment of pancreatic ⁇ -cells with soluble MANF can shift the ER towards an oxidizing environment and may be used to treat or prevent the development of a pancreatic ⁇ cell disorder in a subject.

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